Humanized anti-Tau(pS422) antibodies and methods of use

ABSTRACT

The invention provides humanized anti-human Tau(pS422) antibodies and methods of using the same.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is herein incorporated byreference in its entirety. Said ASCII copy, created on Feb. 1, 2021, isnamed SequenceListing.txt and is 68 KB in size.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and is a continuation of pendingU.S. patent application Ser. No. 15/832,176, filed, Dec. 5, 2017, whichis a divisional of U.S. patent application Ser. No. 15/189,711, filedJun. 22, 2016, now U.S. Pat. No. 9,862,763, issued on Jan. 9, 2018,which in turn claims benefit of European Application No. 15173511.5,filed on Jun. 24, 2015. Each of these applications is herebyincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to humanized anti-Tau(pS422) antibodieswhich specifically bind to a phosphorylated tau fragment of SEQ ID NO:03 and their use for the treatment of brain diseases.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains references to amino acids and/or nucleic acidsequences that have been filed concurrently herewith as sequence listingtext file “0399723.txt”, file size of 68 KB, created on Jun. 21, 2016.The aforementioned sequence listing is hereby incorporated by referencein its entirety pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND OF THE INVENTION

Human tau (microtubule-associated protein tau (neurofibrillary tangleprotein, paired helical filament-tau, PHF-tau)) is a neuronalmicrotubule-associated protein found predominantly in axons andfunctions to promote tubulin polymerization and to stabilizemicrotubules. Eight isoforms (isoform A, B, C, D, E, F, G, fetal-tau)are found in the human brain, the longest isoform comprising 441 aminoacids (isoform F, Uniprot P10636-8). Tau and its properties are alsodescribed by Reynolds, C. H., et al., J. Neurochem. 69 (1997) 191-198.

Tau, in its hyperphosphorylated form, is the major component of pairedhelical filaments (PHF), the building block of neurofibrillary lesionsin Alzheimer's disease (AD) brain. Tau can be phosphorylated at itsserine or threonine residues by several different kinases includingGSK3beta, cdk5, MARK and members of the MAP kinase family.

Tauopathies are characterized by abnormal hyperphosphorylation of tauand are according to Iqbal, K., et al. (Biochim. Biophys. Acta 1739(2005) 198-210):

-   -   Alzheimer disease, including tangle-only form of the disease    -   Down syndrome, adult cases    -   Guam Parkinsonism dementia complex    -   Dementia pugilistica    -   Pick disease    -   Dementia with argyrophilic grains    -   Fronto-temporal dementia    -   Cortico-basal degeneration    -   Pallido-ponto-nigral degeneration    -   Progressive supranuclear palsy    -   Gerstmann-Strässler-Scheinker disease with tangles.

So far nearly 40 serine (S)/threonine (T) phosphorylation sites havebeen found in tau from Alzheimer's disease brains (Hanger, D. P., etal., J. Biol. Chem. 282 (2007) 23645-23654). The development of taupathology in Alzheimer's disease is related to its phosphorylationstate. However, most of the 40 phosphorylation sites are not associatedwith disease pathology since they are also found in tau extracted fromhealthy, fetal brain tissue. Only a few phosphorylations are unique tothe disease state and are presumably responsible for the abnormal,aggregation and characteristic insolubility that define tau in the PHFsof Alzheimer brain (Morishima-Kawashima, M., et al., J. Biol. Chem. 270(1995) 823-829). According to Pei, J. J., et al. (J. Alzheimer's Disease14 (2008) 385-392) the existing literature provides limited and unclearinformation about which of these sites are specific to AD brains. Peiused a list of phospho-specific antibodies to tau and measured theirlevels in homogenates of the medial temporal cortex from 22 AD patientsand 10 controls.

Bussiere, T., et al. (Acta Neuropathol. 97 (1999) 221-230) describedthat phosphorylated serine 422 (pS422) on tau proteins is a pathologicalepitope found in several diseases with neurofibrillary degeneration.Augustinack, J. C., et al., (Acta Neuropathol. 103 (2002) 26-35)described pS422 as correlating with the severity of neuronal pathologyin Alzheimer's disease. Guillozet-Bongaarts, A., (J. Neurochem. 97(2006) 1005-1014) described the phosphorylation of tau at serine 422 asbeing part of the maturation process of PHFs. Tau pS422 is also found inassociation with developing pathology in various transgenic mouse modelsof Alzheimer's disease. Thus, Deters, N., et al., mentioned in Biochem.Biophys. Res. Commun. 379 (2009) 400-405 that double-transgenic Dom5/pR5mice showed 7-fold increased numbers of hippocampal neurons that containtau specifically phosphorylated the pathological 5422 epitope. Goetz,J., et al., (Science 293 (2001) 1491-1495) reported the appearance oftau phosphorylated at S422 in the brains of tau P301L transgenic miceinjected with Abeta42 fibrils.

EP 2 009 104 relates to epitopes of the tau protein which occur in aphosphorylated state in tau protein from Alzheimer's disease PHFs and tothe use of said epitopes for the generation of antibodies specificallydetecting Alzheimer's tau protein. WO 2002/062851 and U.S. Pat. No.7,446,180 relate to antibodies with a specificity to an abnormallytruncated form of tau protein and diagnostic and therapeutic aspects inrelation to Alzheimer's disease and related tauopathies.

WO 1998/22120 relates to a method of treating a patient with Alzheimer'sdisease comprising the step of administering to the patient an antibodyagainst phosphorylated tau fragment of amino acids about 207 to about222, amino acids about 224 to about 240, and amino acids about 390 toabout 408. Animal studies where the phosphorylated tau fragment 379-408[P-Ser396,404] is used to vaccinate tau transgenic mice are mentioned inAsuni, A. A., et al., J. Neuroscience 27 (2007) 9115-9129. US2008/0050383 relates to methods of treating and preventing Alzheimer'sdisease or other tauopathies in a subject by administering a tau proteinfragment.

Hasegawa, M., et al. (FEBS Lett. 384 (1996) 25-30) report monoclonalantibody (AP422) specific for phosphoserine 422 inmicrotubule-associated protein tau.

In WO 2001/55725 an antibody that specifically recognizes tau and anantibody that specifically recognizes phospho-tau (181) for use in amethod for the in vivo diagnosis of a tauopathy and/or for the in vivodifferential diagnosis of a tauopathy versus a non-tauopathy isreported.

In WO 2002/027017 an antibody prepared from a polypeptide immunogenhaving a phosphorylated serine is reported. WO 2002/062851 relates toantibodies with a specificity to an abnormally truncated form of tauprotein and diagnostic and therapeutic aspects in relation toAlzheimer's disease and related tauopathies.

In WO 2004/016655 an antibody specific to a central nervous system (CNS)tau protein, wherein the antibody specifically recognizes a CNS tauprotein but not a peripheral tau protein and wherein the antibodyspecifically recognizes an amino acid sequence of a connective portionbetween the amino acid sequence encoded by Exon 4 of a gene encoding atau protein and the amino acid sequence encoded by Exon 5 thereof as anepitope is reported.

Monoclonal antibodies against Tau(pS422) are described, for example, inEP 1 876 185. Polyclonal antibodies against Tau(pS422) are commerciallyavailable (e.g., ProSci Inc. and Biosource International).

In WO 2006/055178 a method for inhibiting the phosphorylation of tauprotein at Ser202/Thr205 comprising contacting a sample containing a tauprotein with the antibody or antigen binding fragment that binds amyloidbeta-derived diffusible ligands thereby inhibiting the phosphorylationof tau protein at Ser202/Thr205 is reported.

An antibody preparation that specifically binds to tau phosphorylated attyr394 and/or tyr310 is reported in WO 2007/019273. Animal studies wherethe phosphorylated tau fragment 379-408 [P-Ser396,404] is used tovaccinate tau transgenic mice are mentioned in Asuni, A. A. et al., J.Neuroscience 27 (2007) 9115-9129.

EP 2 009 104 relates to epitopes of the tau protein which occur in aphosphorylated state in tau protein from Alzheimer's disease PHFs and tothe use of said epitopes for the generation of antibodies specificallydetecting Alzheimer's tau protein.

US 2008/0050383 relates to methods of treating and preventingAlzheimer's disease or other tauopathies in a subject by administering atau protein fragment.

In WO 2010/037135 an isolated, synthetic or recombinant polypeptide orpeptide comprising a first domain comprising, or consisting of, a ligandfor a blood brain barrier (BBB) receptor or equivalent and a seconddomain comprising, or consisting of an enzyme or composition that slowsthe rate of aggregation of a protein aggregate, inhibits the formationof a protein aggregate, or reverses, digests or dissolves a proteinaggregate is reported. An antibody, particularly a monoclonal antibodyor functional parts thereof, capable of recognizing and binding to a tauprotein in vitro and/or in vivo is reported in WO 2010/115843.

In WO 2011/026031 a monoclonal antibody or its fragment thatspecifically binds tau oligomers and does not bind soluble tau or taufibrils, useful for treating tauopathy, e.g., Alzheimer's disease,progressive supranuclear palsy and corticobasal degeneration isreported. An isolated antibody that specifically binds human tau proteinphosphorylated at one or more of Ser(238) and Thr(245) is reported in WO2011/053565.

In WO 2012/045882 an antibody which specifically binds to aphospho-epitope on the mammalian tau protein, useful for treatingneurodegenerative disorders such as tauopathies, and for treating oralleviating cognitive deficits is reported. A human monoclonal anti-tauantibody or a tau binding fragment thereof is reported in WO2012/049570. A method of preventing or treating Alzheimer's disease orother tauopathies in a subject, comprising administering antibodies to ahuman in need of therapy for Alzheimer's disease or other tauopathy, theantibodies having specificity to abnormal forms of tau protein, saidantibody showing no binding and/or reactivity to a normal tau proteinand being administered under conditions and in an amount(s) effective toprevent or treat Alzheimer's disease or other tauopathy is reported inWO 2012/106363.

In WO 2012/149365 an antibody which shows reactivity with aggregated tauand substantially no reactivity with non-aggregated tau, wherein theaggregated tau comprises at least two tau proteins cross-linked to eachother, either directly or through a linker, at one or more cysteineresidues is reported.

A composition useful in treating tauopathy, e.g., Alzheimer diseasecomprises antibody binding to tau, phosphorylated serine modifiedcompound at specific position specifically binding to specificphosphorylated tau and its fragment and carrier is reported in WO2010/142423.

In EP 1 876 185 A, an antibody which recognizes phosphorylatedpolypeptides is reported. In WO 2013/151762 a humanized tau antibody isreported. In WO 2014/016737 novel chicken monoclonal antibodies againsthuman phosphorylated tau and uses thereof are reported. In WO2014/016737 novel chicken monoclonal antibodies against humanphosphorylated tau and uses thereof are reported. Antibodies selectivefor pathological tau dimers and prefibrillar pathological tau oligomersand their uses in treatment, diagnosis and monitoring of tauopathies arereported in WO 2012/149365.

In WO 2015/091656 humanized anti-Tau(pS422) antibodies and methods ofuse are reported. In WO 2015/101586 bispecific anti-hapten/anti-bloodbrain barrier receptor antibodies, complexes thereof and their use asblood brain barrier shuttles are reported.

BRIEF SUMMARY OF THE INVENTION

The invention provides anti-human Tau(pS422) antibodies, especiallyhumanized anti-human Tau(pS422) antibodies, and methods of using thesame.

The humanized antibodies as reported herein were not available bystandard humanization methods. It was required to introduce non-standardmutations in the amino acid sequence in order to obtain a humanizedantibody with comparable binding characteristics and pharmacokineticproperties as the parent rabbit antibody comprising variable domainswith the amino acid sequence of SEQ ID NO: 07 and SEQ ID NO: 11. This isespecially important as the antibodies as reported herein are intendedto cross the human blood-brain-barrier and to be effective within thehuman brain. Thus, the generally applied criteria for the selection ofhumanized antibodies are not sufficiently stringent in order to beapplied directly in the current case.

One aspect as reported herein is a (humanized) antibody thatspecifically binds to human Tau(pS422), wherein the antibody

-   -   i) specifically binds to a polypeptide that has the amino acid        sequence of SEQ ID NO: 03, and/or    -   ii) does not bind to full length human tau (SEQ ID NO: 01) at 1        μg/mL, and/or    -   iii) specifically binds to full length human tau phosphorylated        at the serine at position 422 (SEQ ID NO: 02), and/or    -   iv) specifically binds to aggregates of human tau phosphorylated        at the serine at position 422 (SEQ ID NO: 02), and/or    -   v) specifically binds to human tau that has the amino acid        sequence of SEQ ID NO: 01 and that has the amino acid mutation        S422A.

The antibodies as reported herein show a selectivity with respect tohuman tau phosphorylated at the serine at position 422, with respect tonot-phosphorylated wild-type human tau and the tau mutant S422A. Thenot-phosphorylated wild-type human tau and the tau mutant S422A are notbound at all or with a lower affinity, respectively.

One aspect as reported herein is a (humanized) anti-human Tau(pS422)monoclonal antibody which is a humanized variant of antibody mAb 086produced by grafting the HVRs of mAb 086 onto human framework andconstant regions, wherein selected human variable region frameworkresidues are optionally substituted as follows:

-   -   a selected human variable region framework residue is        substituted by an equivalent framework amino acid from the        rabbit mAb 086 antibody when the amino acid (1) noncovalently        binds antigen directly, (2) is adjacent to an HVR region, (3)        otherwise interacts with an HVR region, or (4) participates in        the VL-VH interface;    -   or    -   a human framework amino acid that is unusual for a human        immunoglobulin at that position is substituted with an amino        acid from the equivalent position of the rabbit donor antibody        or from the equivalent position of a more typical human        immunoglobulin.

One aspect as reported herein is a (humanized) antibody thatspecifically binds to human Tau(pS422) comprising the amino acidsequence of SEQ ID NO: 02, wherein the antibody has inhibitory activityagainst Tau(pS422)-induced cytotoxicity, and wherein the antibody bindsto a fragment of the human Tau(pS422) protein, and wherein said fragmentcomprises the amino acids residues 416 to 430 of SEQ ID NO: 02, andwherein said antibody comprises a human constant region, wherein theantibody is selected from any of (1) to (2) below;

(1)

-   -   (a) an antibody comprising a heavy chain having the amino acid        sequence of SEQ ID NO: 08 as HVR-H1, the amino acid sequence of        SEQ ID NO: 09 as HVR-H2, and the amino acid sequence of SEQ ID        NO: 10 as HVR-H3, and a light chain having the amino acid        sequence of SEQ ID NO: 70 as HVR-L1, the amino acid sequence of        SEQ ID NO: 72 as HVR-L2, and the amino acid sequence of SEQ ID        NO: 15 as HVR-L3;    -   or    -   (b) an antibody comprising a heavy chain having the amino acid        sequence of SEQ ID NO: 08 as HVR-H1, the amino acid sequence of        SEQ ID NO: 09 as HVR-H2, and the amino acid sequence of SEQ ID        NO: 10 as HVR-H3, and a light chain having the amino acid        sequence of SEQ ID NO: 12 as HVR-L1, the amino acid sequence of        SEQ ID NO: 14 as HVR-L2, and the amino acid sequence of SEQ ID        NO: 74 as HVR-L3;    -   or    -   (c) an antibody comprising a heavy chain having the amino acid        sequence of SEQ ID NO: 08 as HVR-H1, the amino acid sequence of        SEQ ID NO: 09 as HVR-H2, and the amino acid sequence of SEQ ID        NO: 10 as HVR-H3, and a light chain having the amino acid        sequence of SEQ ID NO: 71 as HVR-L1, the amino acid sequence of        SEQ ID NO: 73 as HVR-L2, and the amino acid sequence of SEQ ID        NO: 15 as HVR-L3;    -   or    -   (d) an antibody comprising a heavy chain having the amino acid        sequence of SEQ ID NO: 08 as HVR-H1, the amino acid sequence of        SEQ ID NO: 77 as HVR-H2, and the amino acid sequence of SEQ ID        NO: 10 as HVR-H3, and a light chain having the amino acid        sequence of SEQ ID NO: 12 as HVR-L1, the amino acid sequence of        SEQ ID NO: 14 as HVR-L2, and the amino acid sequence of SEQ ID        NO: 75 as HVR-L3;        (2) an antibody having one or more conservative amino acid        substitutions in the antibody of (1), which has equivalent        activity as the antibody of (1).

One aspect as reported herein is a humanized antibody that specificallybinds to human Tau(pS422), wherein the antibody comprises

-   -   a) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        18 and 10, or    -   b) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        09 and 10, or    -   c) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        77 and 10.

In one preferred embodiment the humanized antibody as reported hereinhas in the light chain variable domain at position 32 a lysine (K) aminoacid residue (numbering according to Kabat).

In one embodiment the humanized antibody comprises

-   -   a) in the light chain variable domain the HVRs of SEQ ID NO: 71,        73 and 15, or    -   b) in the light chain variable domain the HVRs of SEQ ID NO: 70,        72 and 15, or    -   c) in the light chain variable domain the HVRs of SEQ ID NO: 12,        14 and 79, or    -   d) in the light chain variable domain the HVRs of SEQ ID NO: 71,        81 and 15.

In one embodiment the humanized antibody as reported herein comprises

-   -   a) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        18 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 13, 14 and 15, or    -   b) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        09 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 12, 05 and 15, or    -   c) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        09 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 13, 14 and 15, or    -   d) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        09 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 71, 73 and 15, or    -   e) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        09 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 70, 72 and 15, or    -   f) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        77 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 12, 14 and 79, or    -   g) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        09 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 12, 14 and 79, or    -   h) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        77 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 71, 81 and 15.

One preferred aspect is a humanized antibody that specifically binds tohuman Tau(pS422), wherein the antibody comprises the HVRs from VH35H5and VL31A1.

In one embodiment this humanized antibody that specifically binds tohuman Tau(pS422) comprises in the heavy chain variable domain the HVRsof SEQ ID NO: 08, 09 and 10, and in the light chain variable domain theHVRs of SEQ ID NO: 70, 72 and 15. SEQ ID NO: 70 corresponds to thesequence of HVR-L1 and has at position 32 according to Kabat the aminoacid residue lysine.

In one embodiment this this humanized antibody that specifically bindsto human Tau(pS422) comprises a heavy chain variable domain that has theamino acid sequence of SEQ ID NO: 65 and a light chain variable domainthat has the amino acid sequence of SEQ ID NO: 66.

One preferred aspect is a humanized antibody that specifically binds tohuman Tau(pS422), wherein the antibody comprises the HVRs from VH35H5and VL49G1.

In one preferred embodiment this humanized antibody that specificallybinds to human Tau(pS422) comprises in the heavy chain variable domainthe HVRs of SEQ ID NO: 08, 09 and 10, and in the light chain variabledomain the HVRs of SEQ ID NO: 71, 73 and 15. SEQ ID NO: 71 correspondsto the sequence of HVR-L1 and has at position 32 according to Kabat theamino acid residue lysine.

In one embodiment this this humanized antibody that specifically bindsto human Tau(pS422) comprises a heavy chain variable domain that has theamino acid sequence of SEQ ID NO: 65 and a light chain variable domainthat has the amino acid sequence of SEQ ID NO: 67.

One preferred aspect is a humanized antibody that specifically binds tohuman Tau(pS422), wherein the antibody comprises the HVRs from VH35H5and VL35F2.

In one embodiment this humanized antibody that specifically binds tohuman Tau(pS422) comprises in the heavy chain variable domain the HVRsof SEQ ID NO: 08, 09 and 10, and in the light chain variable domain theHVRs of SEQ ID NO: 12, 14 and 74. SEQ ID NO: 12 corresponds to thesequence of HVR-L1 and has at position 32 according to Kabat the aminoacid residue lysine.

In one embodiment this this humanized antibody that specifically bindsto human Tau(pS422) comprises a heavy chain variable domain that has theamino acid sequence of SEQ ID NO: 65 and a light chain variable domainthat has the amino acid sequence of SEQ ID NO: 68.

One preferred aspect is a humanized antibody that specifically binds tohuman Tau(pS422), wherein the antibody comprises the HVRs from VH76A6and VL35G4.

In one embodiment this humanized antibody that specifically binds tohuman Tau(pS422) comprises in the heavy chain variable domain the HVRsof SEQ ID NO: 08, 77 and 10, and in the light chain variable domain theHVRs of SEQ ID NO: 12, 14 and 75. SEQ ID NO: 12 corresponds to thesequence of HVR-L1 and has at position 32 according to Kabat the aminoacid residue lysine.

In one embodiment this this humanized antibody that specifically bindsto human Tau(pS422) comprises a heavy chain variable domain that has theamino acid sequence of SEQ ID NO: 76 and a light chain variable domainthat has the amino acid sequence of SEQ ID NO: 69.

In one embodiment the humanized antibody as reported herein comprises

-   -   a) a heavy chain variable domain of SEQ ID NO: 20 and a light        chain variable domain of SEQ ID NO: 17, or    -   b) a heavy chain variable domain of SEQ ID NO: 19 and a light        chain variable domain of SEQ ID NO: 16, or    -   c) a heavy chain variable domain of SEQ ID NO: 19 and a light        chain variable domain of SEQ ID NO: 17, or    -   d) a heavy chain variable domain of SEQ ID NO: 21 and a light        chain variable domain of SEQ ID NO: 17, or    -   e) a heavy chain variable domain of SEQ ID NO: 65 and a light        chain variable domain of SEQ ID NO: 67, or    -   f) a heavy chain variable domain of SEQ ID NO: 65 and a light        chain variable domain of SEQ ID NO: 66, or    -   g) a heavy chain variable domain of SEQ ID NO: 76 and a light        chain variable domain of SEQ ID NO: 78, or    -   h) a heavy chain variable domain of SEQ ID NO: 19 and a light        chain variable domain of SEQ ID NO: 78, or    -   i) a heavy chain variable domain of SEQ ID NO: 76 and a light        chain variable domain of SEQ ID NO: 80.

In one preferred embodiment the humanized antibody comprises a heavychain variable domain of SEQ ID NO: 65 and a light chain variable domainof SEQ ID NO: 66 or 67 or 68.

In one preferred embodiment the humanized antibody comprises a heavychain variable domain of SEQ ID NO: 76 and a light chain variable domainof SEQ ID NO: 69.

In one embodiment the humanized antibody comprises a heavy chainvariable domain of SEQ ID NO: 76 or 19 and a light chain variable domainof SEQ ID NO: 78.

In one embodiment the antibody is for use in the treatment ofAlzheimer's disease.

In one embodiment the antibody is effector function silent. In oneembodiment the antibody has no effector function. In one embodiment theantibody is of the human IgG1 subclass and has the mutations L234A,L235A and P329G in both heavy chains (numbering according to the EUindex of Kabat).

In one embodiment the antibody

-   -   i) specifically binds to a polypeptide that has the amino acid        sequence of SEQ ID NO: 03, and/or    -   ii) does not bind to full length human tau (SEQ ID NO: 01) at 1        μg/mL, and/or    -   iii) specifically binds to full length human tau phosphorylated        at the serine at position 422 (SEQ ID NO: 02), and/or    -   iv) specifically binds to aggregates of human tau phosphorylated        at the serine at position 422 (SEQ ID NO: 02).

In one embodiment the antibody has an EC₅₀ value for

-   -   a) the human Tau(pS422) fragment that has the amino acid        sequence of SEQ ID NO: 03 of 6 ng/mL or less, and/or    -   b) the full length human Tau(pS422) that has the amino acid        sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or    -   c) aggregates of human Tau(pS422) that has the amino acid        sequence of SEQ ID NO: 02 of 30 ng/mL or less, and/or    -   d) the human tau that has the amino acid sequence of SEQ ID NO:        01 and that has the amino acid mutation S422A of 125 ng/mL or        less.

In one embodiment the antibody specifically binds to human Tau(pS422)(SEQ ID NO: 02) and does not bind to human tau (SEQ ID NO: 01).

In one embodiment the antibody is a monoclonal antibody.

In one embodiment the antibody is an antibody fragment that binds tohuman Tau(pS422) and

-   -   i) specifically binds to a polypeptide that has the amino acid        sequence of SEQ ID NO: 03, and/or    -   ii) does not bind to full length human tau (SEQ ID NO: 01) at 1        μg/mL, and/or    -   iii) specifically binds to full length human tau phosphorylated        at the serine at position 422 (SEQ ID NO: 02), and/or    -   iv) specifically binds to aggregates of human tau phosphorylated        at the serine at position 422 (SEQ ID NO: 02), and/or    -   v) specifically binds to full length human tau that has the        amino acid sequence of SEQ ID NO: 01 and that has the amino acid        mutation S422A, and/or    -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that has        the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or less,        and/or    -   vii) has an EC₅₀ value for the full length human Tau(pS422) that        has the amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or        less, and/or    -   viii) has an EC₅₀ value for aggregates of human Tau(pS422) that        has the amino acid sequence of SEQ ID NO: 02 of 30 ng/mL or        less, and/or    -   ix) has an EC₅₀ value for the human tau that has the amino acid        sequence of SEQ ID NO: 01 and that has the amino acid mutation        S422A of 125 ng/mL or less.

In one embodiment the antibody is

-   -   a) a full length antibody of the human subclass IgG1, or    -   b) a full length antibody of the human subclass IgG4, or    -   c) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A and P329G,    -   d) a full length antibody of the human subclass IgG4 with the        mutations S228P, L235E and P329G,    -   e) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A and P329G in both heavy chains and the        mutations T366W and S354C in one heavy chain and the mutations        T366S, L368A, Y407V and Y349C in the respective other heavy        chain, or    -   f) a full length antibody of the human subclass IgG4 with the        mutations S228P and

P329G in both heavy chains and the mutations T366W and S354C in oneheavy chain and the mutations T366S, L368A, Y407V and Y349C in therespective other heavy chain.

One aspect as reported herein is a (humanized) anti-human Tau(pS422)antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 08,            SEQ ID NO: 18 and SEQ ID NO: 10,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 13,            SEQ ID NO: 14 and SEQ ID NO: 15,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One aspect as reported herein is a (humanized) anti-human Tau(pS422)antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 08,            SEQ ID NO: 09 and SEQ ID NO: 10,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 12,            SEQ ID NO: 05 and SEQ ID NO: 15,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One aspect as reported herein is a (humanized) anti-human Tau(pS422)antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 08,            SEQ ID NO: 09 and SEQ ID NO: 10,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 13,            SEQ ID NO: 14 and SEQ ID NO: 15,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or    -   iv) specifically binds to aggregates of human tau phosphorylated        at the serine at position 422 (SEQ ID NO: 02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One preferred aspect as reported herein is a (humanized) anti-humanTau(pS422) antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 08,            SEQ ID NO: 09 and SEQ ID NO: 10,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 71,            SEQ ID NO: 73 and SEQ ID NO: 15,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or    -   iv) specifically binds to aggregates of human tau phosphorylated        at the serine at position 422 (SEQ ID NO: 02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or    -   vii) has an EC₅₀ value for the full length human Tau(pS422) that        has the amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or        less, and/or    -   viii) has an EC₅₀ value for aggregates of human Tau(pS422) that        has the amino acid sequence of SEQ ID NO: 02 of 30 ng/mL or        less, and/or    -   ix) has an EC₅₀ value for the human tau that has the amino acid        sequence of SEQ ID NO: 01 and that has the amino acid mutation        S422A of 125 ng/mL or less.

One preferred aspect as reported herein is a (humanized) anti-humanTau(pS422) antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 08,            SEQ ID NO: 09 and SEQ ID NO: 10,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 70,            SEQ ID NO: 72 and SEQ ID NO: 15,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One aspect as reported herein is a (humanized) anti-human Tau(pS422)antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 08,            SEQ ID NO: 09 and SEQ ID NO: 10,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 12,            SEQ ID NO: 14 and SEQ ID NO: 79,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One preferred aspect as reported herein is a (humanized) anti-humanTau(pS422) antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 08,            SEQ ID NO: 09 and SEQ ID NO: 10,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 12,            SEQ ID NO: 14 and SEQ ID NO: 74,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One preferred aspect as reported herein is a (humanized) anti-humanTau(pS422) antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 08,            SEQ ID NO: 77 and SEQ ID NO: 10,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 12,            SEQ ID NO: 14 and SEQ ID NO: 75,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One aspect as reported herein is a (humanized) anti-human Tau(pS422)antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 20,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 17,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One aspect as reported herein is a (humanized) anti-human Tau(pS422)antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 19,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 16,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One aspect as reported herein is a (humanized) anti-human Tau(pS422)antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 19,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 17,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One aspect as reported herein is a (humanized) anti-human Tau(pS422)antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 21,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 17,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One preferred aspect as reported herein is a (humanized) anti-humanTau(pS422) antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 65,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 67,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One preferred aspect as reported herein is a (humanized) anti-humanTau(pS422) antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 65,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 66,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One preferred aspect as reported herein is a (humanized) anti-humanTau(pS422) antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 65,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 68,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One aspect as reported herein is a (humanized) anti-human Tau(pS422)antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 19,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 78,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

One preferred aspect as reported herein is a (humanized) anti-humanTau(pS422) antibody, characterized in that

-   -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 76,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue or the glycine-lysine            dipeptide can be present or absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 69,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

In one preferred embodiment of all aspects the anti-human Tau(pS422)antibody is characterized in that the antibody has in the light chainvariable domain at position 32, a lysine (K) amino acid residue(numbering according to Kabat).

In one preferred embodiment of all aspects the anti-human Tau(pS422)antibody is characterized in that the antibody has in the heavy chainvariable domain at positions 4, 24 and 78, a valine residue.

In one preferred embodiment of all aspects the anti-human Tau(pS422)antibody is characterized in that the antibody has in the heavy chainvariable domain at position 71, an arginine residue.

One aspect as reported herein is an isolated nucleic acid encoding a(humanized) antibody as reported herein. This nucleic acid comprises anucleic acid encoding the antibody heavy chain and one nucleic acidencoding the antibody light chain. This nucleic acid comprises at leastone expression cassette. Preferably said nucleic acid comprises twoexpression cassettes.

One aspect as reported herein is a host cell comprising the nucleic acidas reported herein.

One aspect as reported herein is a method of producing a (humanized)antibody comprising the steps of culturing the host cell as reportedherein so that the antibody is produced.

In one embodiment the method further comprises the step of recoveringthe antibody from the cell or the cultivation medium.

One aspect as reported herein is a pharmaceutical formulation comprisingthe (humanized) antibody as reported herein and a pharmaceuticallyacceptable carrier.

In one embodiment the pharmaceutical formulation further comprises anadditional therapeutic agent.

In one embodiment the additional therapeutic agent is an anti-amyloidtherapeutic agent. In one embodiment the anti-amyloid therapeutic agentis an anti-human alpha-synuclein antibody or an anti-Abeta antibody.

One aspect as reported herein is the (humanized) antibody as reportedherein for use as a medicament.

One aspect as reported herein is the (humanized) antibody as reportedherein for use in the treatment of Alzheimer's disease.

One aspect as reported herein is the (humanized) antibody as reportedherein for use in the treatment of prodromal Alzheimer's disease.

One aspect as reported herein is the (humanized) antibody as reportedherein for use in the treatment of mild Alzheimer's disease.

One aspect as reported herein is the (humanized) antibody as reportedherein for use in reducing Tau(pS422)-induced neurodegeneration.

One aspect as reported herein is the (humanized) antibody as reportedherein for use in maintaining cognition and function.

One aspect as reported herein is the (humanized) antibody as reportedherein for use in slowing the rate of cognitive and functional decline.

One aspect as reported herein is the (humanized) antibody as reportedherein for use in slowing down the rate of neurofibrillary tangleaccumulation.

In one embodiment of the previous aspects the use is by reducingneurofibrillary tangle burden by clearing Tau(pS422).

In one embodiment of the previous aspects the use is by preventingneurofibrillary tangle build up.

In one embodiment of the previous aspects the use is byremoving/clearing neurofibrillary tangles.

In one embodiment the preventing and/or removing is by promoting theintracellular clearance of tau aggregates.

In one embodiment of the previous aspects the use is by inhibitingneurofibrillary tangle spreading. In one embodiment the inhibiting is bypreventing interneuronal transfer of pathological tau forms/seeds.

Aspects of the current invention are also methods of treatmentcomprising administering the (humanized) antibody as reported herein fortreating Alzheimer's disease, for treating prodromal Alzheimer'sdisease, for treating mild Alzheimer's disease, for reducingTau(pS422)-induced neurodegeneration, for maintaining cognition andfunction, for slowing the rate of cognitive and functional decline,and/or for slowing down the rate of neurofibrillary tangle accumulation.

One aspect as reported herein is the use of the (humanized) antibody asreported herein in the manufacture of a medicament.

In one embodiment the medicament is for treatment of Alzheimer'sdisease.

In one embodiment the medicament is for treatment of prodromalAlzheimer's disease.

In one embodiment the medicament is for treatment of mild Alzheimer'sdisease.

In one embodiment the medicament is for reducing Tau(pS422)-inducedneurodegeneration.

In one embodiment the medicament is for maintaining cognition andfunction.

In one embodiment the medicament is for slowing the rate of cognitiveand functional decline.

One aspect as reported herein is a method of treating an individualhaving Alzheimer's disease comprising administering to the individual aneffective amount of a (humanized) anti-human Tau(pS422) antibody asreported herein.

One aspect as reported herein is a method of reducing Tau(pS422)-inducedneurodegeneration in an individual comprising administering to theindividual an effective amount of a (humanized) anti-human Tau(pS422)antibody as reported herein to reduce Tau(pS422)-inducedneurodegeneration.

One aspect as reported herein is a method of maintaining cognition andfunction in an individual comprising administering to the individual aneffective amount of a (humanized) anti-human Tau(pS422) antibody asreported herein to maintain cognition and function.

One aspect as reported herein is a method of slowing the rate ofcognitive and functional decline in an individual comprisingadministering to the individual an effective amount of a (humanized)anti-human Tau(pS422) antibody as reported herein to slow the rate ofcognitive and functional decline.

One aspect as reported herein is the use of a (humanized) anti-humanTau(pS422) antibody as reported herein in the reduction ofTau(pS422)-induced neurodegeneration.

One aspect as reported herein is the use of a (humanized) anti-humanTau(pS422) antibody as reported herein in maintaining cognition andfunction.

One aspect as reported herein is the use of a (humanized) anti-humanTau(pS422) antibody as reported herein in slowing the rate of cognitiveand functional decline.

The antibodies as reported herein can be used in the treatment ofAlzheimer's disease.

With the (humanized) antibodies as reported herein inhibition/reductionof progression of Alzheimer's disease and neuropathology can beeffected.

The (humanized) antibodies as reported herein can be used to protect ananimal from the development of Alzheimer's disease or even used to stopthe progression of Alzheimer's disease in an animal. In one embodimentthe animal is a human.

In one embodiment the (humanized) antibody as reported herein binds toTau(pS422) on brain sections of Tau(pS422) transgenic mice andAlzheimer's disease patients; and/or labels Tau(pS422) in Tau(pS422)transgenic cells.

The (humanized) antibodies as reported herein can be used for thetreatment of Alzheimer's disease.

One aspect as reported herein is a (humanized) antibody thatspecifically binds to the amino acid sequence of SEQ ID NO: 03 in humanTau(pS422).

The (humanized) antibodies as reported herein specifically bindto/recognize early and late stage disease-relevant forms of humanTau(pS422).

One aspect as reported herein is the use of the (humanized) antibody asreported herein for the prevention of human Tau(pS422)-relatedAlzheimer's disease spread.

One aspect as reported herein is the use of the (humanized) antibody asreported herein for the reduction of lysosomal membrane disintegration.

One aspect as reported herein is the use of the (humanized) antibody asreported herein for the stabilization of lysosome membrane against humanTau(pS422)-induced destabilization and/or disintegration.

One aspect as reported herein is the use of the (humanized) antibody asreported herein for the prevention of Alzheimer's disease progression.

The (humanized) antibodies as reported herein function by antibodymediated inhibition of human Tau(pS422) seeding and spreading betweencells.

The (humanized) antibodies as reported herein protect lysosomes fromfibrillar damage by binding to human Tau(pS422).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of a pharmacokinetic study in cynomolgus; soliddiamond: VH35H5/VL31A1; open square: VH35H5/VL35F2; solid square:VH76A6/VL35G4; solid triangle: VH32/VL22; solid circle: VH00/VL00;X-axis: time after dosing [h]; y-axis: Serum concentration, dose-norm.[(μg/mL)/(mg/kg)].

FIG. 2 shows the zoom to time range 0-200 h of FIG. 1 .

FIG. 3A-FIG. 3D show biochemical binding of different combinations ofhumanized VH and VL to (FIG. 3A) phosphorylated tau peptide, (FIG. 3B)phosphorylated full-length human tau, (FIG. 3C) non-phosphorylated taupeptide, (FIG. 3D) non-phosphorylated full-length human tau;(1)=VH00/VL00, (2)=VH32/VL21, (3)=VH20/VL22, (4)=VH32/VL22,(5)=VH33/VL22; coating concentrations: phosphorylated tau peptide: 50ng/ml, all other targets: 1 μg/ml; (comparable results are obtained ifphosphorylated tau peptide is coated with 1 μg/ml (data not shown)).

FIG. 4A-FIG. 4B show biochemical binding of different combinations ofhumanized VH and VL to (FIG. 4A)=full length human tau S422A mutant,(FIG. 4B)=aggregated human Tau(pS422); (1)=VH00/VL00, (2)=VH32/VL21,(3)=VH20/VL22, (4)=VH32/VL22, (5)=VH33/VL22; coating concentrations:phosphorylated tau peptide: 50 ng/ml, all other targets: 1 μg/ml;(comparable results are obtained if phosphorylated tau peptide is coatedwith 1 μg/ml (data not shown)).

FIG. 5 is a Western Blot showing the selectivity of selected humanizedVH/VL combinations; (1)=VH00/VL00, (2)=VH32/VL21, (3)=VH20/VL22,(4)=VH32/VL22, (5)=VH33/VL22.

FIG. 6 shows binding to hyperphosphorylated tau in brain extracts ofAlzheimer's disease patients; (1)=VH00/VL00, (2)=VH32/VL21,(3)=VH32/VL22.

FIG. 7A-FIG. 7B show quantitation of Tau(pS442) levels in mouse brainextracts; max-min, median and mean (+); significance was calculatedusing Student's t-test, *p<0.05, **p<0.01, ***p<0.001. FIG. 7A: x-axis:Tau(pS422) [ng/mg brain]; y-axis: 1: baseline, 2: vehicle, 3:anti-Tau(pS422) antibody (VH35H5/VL31A1) @ 1.7 mg/kg, 4: anti-Tau(pS422)antibody (VH35H5/VL31A1) @ 5 mg/kg, 5: anti-Tau(pS422) antibody(VH35H5/VL31A1) @ 15 mg/kg.

FIG. 7B: x-axis: Tau(pS422) [ng/mg brain]; y-axis: 1: baseline, 2:vehicle, 3: anti-Tau(pS422) antibody (VH35H5/VL49G1) @ 1.7 mg/kg, 4:anti-Tau(pS422) antibody (VH35H5/VL49G1) @ 5 mg/kg, 5: anti-Tau(pS422)antibody (VH35H5/VL49G1) @ 15 mg/kg.

FIG. 8A-FIG. 8B show quantitation of total tau in mouse brain extracts;max-min, median and mean (+). FIG. 8A: x-axis: total tau [ng/mg brain];y-axis: 1: baseline, 2: vehicle, 3: anti-Tau(pS422) antibody(VH35H5/VL49G1) @ 1.7 mg/kg, 4: anti-Tau(pS422) antibody (VH35H5/VL49G1)@ 5 mg/kg, 5: anti-Tau(pS422) antibody (VH35H5/VL49G1) @ 15 mg/kg.

FIG. 8B: x-axis: total tau [ng/mg brain]; y-axis: 1: baseline, 2:vehicle, 3: anti-Tau(pS422) antibody (VH35H5/VL31A1) @ 1.7 mg/kg, 4:anti-Tau(pS422) antibody (VH35H5/VL31A1) @ 5 mg/kg, 5: anti-Tau(pS422)antibody (VH35H5/VL31A1) @ 15 mg/kg.

FIG. 9A-FIG. 9B show quantitative IHC analysis of Tau(pS422) pathologyin anti-Tau(pS422) antibody (VH35H5/VL31A1)-treated TauPS2APP mice;max-min, median and mean (+), significance was calculated usingStudent's t-test, **p<0.01, ***p<0.001. FIG. 9A: Cortex; x-axis:Tau(pS422) area occupancy [%]; y-axis: 1: baseline, 2: vehicle, 3:anti-Tau(pS422) antibody (VH35H5/VL31A1) @ 1.7 mg/kg, 4: anti-Tau(pS422)antibody (VH35H5/VL31A1) @ 5 mg/kg, 5: anti-Tau(pS422) antibody(VH35H5/VL31A1) @ 15 mg/kg. FIG. 9B: Hippocampus; x-axis: area occupancy[%]; y-axis: 1: baseline, 2: vehicle, 3: anti-Tau(pS422) antibody(VH35H5/VL31A1) @ 1.7 mg/kg, 4: anti-Tau(pS422) antibody (VH35H5/VL31A1)@ 5 mg/kg, 5: anti-Tau(pS422) antibody (VH35H5/VL31A1) @ 15 mg/kg.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 01 human tau protein isoform F (441 residues) SEQ ID NO: 02human tau protein isoform F (441 residues)phosphorylated at the serine residue at position 422 SEQ ID NO: 03fragment of human tau protein (residues 416 to 430 of SEQ ID NO: 01)with phosphorylated serine at position 7(corresponding to position 422 of SEQ ID NO: 01):Ser-Ile-Asp-Met-Val-Asp-Ser(PO₃H₂)- Pro-Gln-Leu-Ala-Thr-Leu-Ala-AspSEQ ID NO: 04 rabbit antibody 086 CDRL1-QSSQSVRTNKLA SEQ ID NO: 05rabbit antibody 086 CDRL2-SASTLDF SEQ ID NO: 06rabbit antibody 086 CDRL3-LGYFDCSIADCVA SEQ ID NO: 07rabbit antibody 086 VL00 SEQ ID NO: 08 rabbit antibody 086 CDRH1-SNAINSEQ ID NO: 09 rabbit antibody 086 CDRH2-YIAVSGNTYYASWAKG SEQ ID NO: 10rabbit antibody 086 CDRH3-SNI SEQ ID NO: 11 rabbit antibody 086 VH00SEQ ID NO: 12 humanized CDRL1 variant 1-RSSQSVRTNKLA SEQ ID NO: 13humanized CDRL1 variant 2-RSSQSVRTNRLA SEQ ID NO: 14humanized CDRL2 variant 1-SASTLDY SEQ ID NO: 15humanized CDRL3 variant 1-LGYFDSSADIVA SEQ ID NO: 16humanized VL variant 1-VL21 SEQ ID NO: 17 humanized VL variant 2-VL22SEQ ID NO: 18 humanized CDRH2-YIAVSGNTYYADSVKG SEQ ID NO: 19humanized VH variant 1-VH32 SEQ ID NO: 20 humanized VH variant 2-VH20SEQ ID NO: 21 humanized VH variant 3-VH33 SEQ ID NO: 22humanized CDRL2 variant 2-SASTLQS SEQ ID NO: 23humanized CDRL2 variant 3-SASTLES SEQ ID NO: 24humanized CDRL3 variant 2-LGYFDSSIADSVA SEQ ID NO: 25humanized CDRL3 variant 3-LGYFDSSIADRVA SEQ ID NO: 26humanized CDRL3 variant 4-LGYFDPSIADPVA SEQ ID NO: 27humanized CDRL3 variant 5-LGYFDSSIADIVA SEQ ID NO: 28humanized CDRL3 variant 6-LGYFDPSADPIA SEQ ID NO: 29humanized CDRL3 variant 7-LGYFDPSADPVA SEQ ID NO: 30humanized CDRL1 variant 3-RASQGVRTNKLA SEQ ID NO: 31humanized CDRL1 variant 4-RASQSVRTNKLA SEQ ID NO: 32humanized VL variant 4-VL01 SEQ ID NO: 33 humanized VL variant 5-VL09SEQ ID NO: 34 humanized VL variant 6-VL12 SEQ ID NO: 35humanized VL variant 7-VL15 SEQ ID NO: 36 humanized VL variant 8-VL16SEQ ID NO: 37 humanized VL variant 9-VL17 SEQ ID NO: 38humanized VL variant 10-VL19 SEQ ID NO: 39 humanized VL variant 11-VL28SEQ ID NO: 40 humanized VL variant 12-VL33 SEQ ID NO: 41humanized VL variant 13-VL35 SEQ ID NO: 42 humanized VL variant 14-VL39SEQ ID NO: 43 humanized VL variant 15-VL40 SEQ ID NO: 44humanized VL variant 16-VL41 SEQ ID NO: 45 humanized VL variant 17-VL42SEQ ID NO: 46 humanized VH variant 4-VH01 SEQ ID NO: 47humanized VH variant 5-VH02 SEQ ID NO: 48 humanized VH variant 6-VH03SEQ ID NO: 49 humanized VH variant 7-VH04 SEQ ID NO: 50humanized VH variant 8-VH14 SEQ ID NO: 51 humanized VH variant 9-VH15SEQ ID NO: 52 humanized VH variant 10-VH18 SEQ ID NO: 53humanized VH variant 11-VH19 SEQ ID NO: 54 humanized VH variant 12-VH22SEQ ID NO: 55 humanized VH variant 13-VH23 SEQ ID NO: 56humanized VH variant 14-VH24 SEQ ID NO: 57 humanized VH variant 15-VH31SEQ ID NO: 65 humanized VH variant 16-VH35H5 SEQ ID NO: 66humanized VL variant 18-VL31A1 SEQ ID NO: 67humanized VL variant 19-VL49G1 SEQ ID NO: 68humanized VL variant 20-VL35F2 SEQ ID NO: 69humanized VL variant 21-VL53A2 SEQ ID NO: 70 humanized CDRL1 variant 5SEQ ID NO: 71 humanized CDRL1 variant 6 SEQ ID NO: 72humanized CDRL2 variant 4 SEQ ID NO: 73 humanized CDRL2 variant 5SEQ ID NO: 74 humanized CDRL3 variant 8 SEQ ID NO: 75humanized CDRL3 variant 9 SEQ ID NO: 76 humanized VH variant 17-VH76A6SEQ ID NO: 77 humanized CDRH1 variant 1 SEQ ID NO: 78humanized VL variant 22-VL35G4 SEQ ID NO: 79 humanized CDRL3 variant 10SEQ ID NO: 80 humanized VL variant 23-VL145B12 SEQ ID NO: 81humanized CDRL2 variant 6 SEQ ID NO: 82anti-transferrin receptor antibody heavy chain 1 SEQ ID NO: 83anti-transferrin receptor antibody heavy chain 2 SEQ ID NO: 84anti-transferrin receptor antibody heavy chain 3 SEQ ID NO: 85anti-transferrin receptor antibody light chain SEQ ID NO: 86humanized VL variant 24-VL4G1 SEQ ID NO: 87 humanized CDRL3 variant 11

Sequence correspondence table: variable domain CDR1 CDR2 CDR3 completesequence VL00 04 05 06 07 VL01 04 05 06 32 VL4G1 12 05 87 86 VL09 31 2306 33 VL12 30 22 06 34 VL15 30 22 24 35 VL16 30 22 25 36 VL17 12 05 0637 VL19 12 05 06 38 VL21 12 05 15 16 VL22 13 14 15 17 VL28 13 05 29 39VL31A1 70 72 15 66 VL33 13 05 27 40 VL35 13 05 27 41 VL35F2 12 14 74 68VL35G4 12 14 79 78 VL39 13 05 26 42 VL40 13 05 29 43 VL41 13 05 28 44VL42 13 05 28 45 VL49G1 71 73 15 67 VL53A2 12 14 75 69 VL145B12 71 81 1580 VH00 08 09 10 11 VH01 08 09 10 46 VH02 08 09 10 47 VH03 08 09 10 48VH04 08 09 10 49 VH14 08 09 10 50 VH15 08 09 10 51 VH18 08 18 10 52 VH1908 18 10 53 VH20 08 18 10 20 VH22 08 18 10 54 VH23 08 18 10 55 VH24 0818 10 56 VH31 08 09 10 57 VH32 08 09 10 19 VH33 08 09 10 21 VH35H5 8 910 65 VH76A6 8 77 10 76

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a binding peptide or protein or afunctional part thereof, particularly to an antibody, particularly amonoclonal antibody or a functional part thereof, particularly a bindingpeptide or antibody, which binding peptide or antibody recognizes andspecifically binds to a phospho-epitope on a mammalian, particularly onthe human tau protein or on a fragment thereof, particularly to apathological protein tau conformer, but, in one embodiment, does notbind to the corresponding unphosphorylated epitope and/or to non-relatedepitopes wherein said binding peptide or antibody binds to an epitope ona mammalian, particularly on the human tau protein as shown in SEQ IDNO: 02, consisting of tau amino acid residues 416-430 comprising aphosphorylated Ser at position 422 (pS422).

I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-human Tau(pS422) antibody” and “an antibody thatspecifically binds to human Tau(pS422)” refer to an antibody that iscapable of binding human Tau(pS422) with sufficient affinity such thatthe antibody is useful as a diagnostic and/or therapeutic agent intargeting human Tau(pS422). In one embodiment, the extent of binding ofan anti-human Tau(pS422) antibody to an unrelated, non-human Tau(pS422)protein is less than about 10% of the binding of the antibody to humanTau(pS422) as measured, e.g., by a radioimmunoassay (RIA).

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab)₂;diabodies; linear antibodies; single-chain antibody molecules (e.g.,scFv); and multispecific antibodies formed from antibody fragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. In one embodiment an antibody bindingto the same epitope as a reference antibody blocks binding of thereference antibody to its antigen by 50% or more. In one embodiment anantibody binding to the same epitope as a reference antibody blocksbinding of the reference antibody to its antigen by 80% or more. In oneembodiment an antibody binding to the same epitope as a referenceantibody blocks binding of the reference antibody to its antigen by 90%or more. In one embodiment an antibody binding to the same epitope as areference antibody blocks binding of the reference antibody to itsantigen by 95% or more. In one preferred embodiment an antibody bindingto the same epitope as a reference antibody has binding interactionswith the same residues as the reference antibody on the antigen.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

“Effector functions” refer to those biological activities attributableto the Fc-region of an antibody, which vary with the antibody class.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc-region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc-regions andvariant Fc-regions. In one embodiment, a human IgG heavy chain Fc-regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc-regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc-region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991), NIH Publication 91-3242.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc-region as defined herein. Theterm “full length antibody” denotes a multimeric polypeptide consistingof two antibody light chain polypeptides and two antibody heavy chainpolypeptides linked by disulfide bonds wherein in the two antibody heavychain polypeptides the C-terminal lysine residue (K) can be present ornot.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Bethesda Md. (1991), NIH Publication 91-3242, Vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and formstructurally defined loops (“hypervariable loops”), and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3).

HVRs herein include:

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987)        901-917);    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat, E. A. et al., Sequences of Proteins of Immunological        Interest, 5th ed. Public Health Service, National Institutes of        Health, Bethesda, Md. (1991), NIH Publication 91-3242);    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)); and    -   (d) combinations of (a), (b), and/or (c), including HVR amino        acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),        26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102        (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s).

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-human Tau(pS422) antibody”refers to one or more nucleic acid molecules encoding antibody heavy andlight chains (or fragments thereof), including such nucleic acidmolecule(s) in a single vector or separate vectors, and such nucleicacid molecule(s) present at one or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “human Tau(pS422)”, as used herein, refers to native humanTau(pS422) (UniProt P37840). The term encompasses “full-length”,unprocessed human Tau(pS422) as well as any form of human Tau(pS422)that results from processing in the cell. The term also encompassesnaturally occurring variants of human Tau(pS422), e.g., mutants, splicevariants or allelic variants. The amino acid sequence of humanTau(pS422) is shown in SEQ ID NO: 02.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt,T. J. et al. Kuby Immunology, 6th ed., W. H. Freeman and Co., N.Y.(2007), page 91) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano, S. et al., J.Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991)624-628).

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

II. Compositions and Methods

A. Exemplary Humanized Anti-Human Tau(pS422) Antibodies

The humanized antibodies as reported herein were not available bystandard humanization methods. It was required to introduce non-standardmutations in the amino acid sequence in order to obtain a humanizedantibody with comparable binding characteristics and pharmacokineticproperties as the parent rabbit antibody. This is especially importantas the antibodies as reported herein are intended to cross the humanblood-brain-barrier and to be effective within the human brain. Thus,the generally applied criteria for the selection of humanized antibodieswere not sufficiently stringent in order to be applied directly in thecurrent case.

It has been found that in order to obtain a suitable and developablehumanized antibody two cysteines forming a disulfide-bridge in the CDRL3(light chain CDR3) had to be replaced by serine and isoleucine,respectively. In addition, to ensure proper orientation of the sameCDRL3 an isoleucine residue present in the middle of the rabbit CDRL3was deleted resulting in a humanized CDRL3 that is one amino acidresidue smaller than the parent rabbit CDRL3.

It has also been found that to maintain in vivo pharmacokineticproperties the amino acid residue at position 32 in the light chainshould be a lysine (numbering according to Kabat). The removal of thedisulfide bridge in the light chain HVR-L3 does not have an impact onthe kinetic behavior in vivo.

It has further been found that it is advantageous to maintain threevaline amino acid residues in the heavy chain at positions 4, 24 and 78(numbering according to Kabat). Without being bound by this theory it isassumed that these residues are required to ensure proper presentationof the antigen binding loops of the heavy chain variable region.Additionally the presence of an arginine residue at position 71 in theheavy chain variable domain is advantageous (numbering according toKabat).

The HVR-L3 comprises two aspartic acid residues which might be adeamidation hotspot, especially during prolonged storage. In the lightchain variable domain variant VL35G4 one of these two aspartic acidresidues has been changed.

All numbering as used herein is based on the Kabat variable domainnumbering scheme.

In the following Table characteristics of the different humanizedvariants of the rabbit light chain variable domain in combination withthe humanized heavy chain variable domains VH14 and VH20, respectively,are shown. The binding partner was human Tau(pS422).

ka [1/Ms] kd [1/s] KD [M] t/2 diss [min] T [° C.] VH14 with VL001.04E−03 11 25 VL01 3.82E−03 3 25 VL09 2.35E−03 5 25 VL12 2.48E−03 5 25VL15 3.63E−03 3 25 VL16 n.d. VL17 2.39E−03 5 25 VL17 3.03E−03 4 25 VL191.98E−03 6 25 VL21 2.93E−03 4 25 VL22 3.30E−03 4 25 VL28 3.84E−03 3 25VL33 1.02E−02 1 25 VL35 1.10E−02 1 25 VL39 5.22E−03 2 25 VL40 3.01E−03 425 VL41 n.d. VL42 n.d. VH20 with VL00 n.d. VL01 n.d. VL09 2.14E−03 5 25VL12 n.d. VL15 n.d. VL16 n.d. VL17 5.35E−04 22 25 VL19 3.66E−04 32 25VL19 1.94E+04 1.13E−03 5.84E−8  10.2 37 VL21 7.88E−04 15 25 VL213.03E+04 2.10E−03 6.95E−08 5.5 37 VL22 8.39E−04 14 25 VL22 3.44E+042.37E−03 6.90E−08 4.9 37 VL28 1.27E−03 9 25 VL28 2.50E+04 3.61E−031.45E−07 3.2 37 VL33 1.61E−03 7 25 VL35 1.59E−03 7 25 VL39 1.91E−03 6 25VL40 9.98E−04 12 25 VL41 4.29E−03 3 25 VL42 4.57E−03 3 25

Reference values VH00 with VL00 (rabbit antibody):

-   -   25° C.: kd=2.6E−04; t/2=44 minutes    -   37° C.: ka=3.7E+04, kd=5.25E−03, KD=1.4E−08, t/2=22 minutes

In the following Table characteristics of the different humanizedvariants of the rabbit light chain variable domain in combination withthe humanized light chain variable domains VL17 and VL19, respectively,are shown.

ka [1/Ms] kd[1/s] KD [M] t/2 diss [min] T [° C.] VL17 with VH004.98E−04  23 25 VH01 2.3E−03 5 25 VH02 3.71E−03  3 25 VH03 3.93E−03  325 VH04 4.16E−03  3 25 VH14 3.0E−03 4 25 VH15 3.26E−03  4 25 VH182.3E−03 5 25 VH19 n.d. VH20 5.4E−04 22 25 VH22 2.0E−03 6 25 VH23 7.0E−0417 25 VH24 7.9E−04 15 25 VH31 n.d. VH32 n.d. VH33 n.d. VL19 with VH00n.d. VH01 1.9E−03 6 25 VH02 n.d. VH03 n.d. VH04 n.d. VH14 2.0E−03 6 25VH15 n.d. VH18 1.9E−03 6 25 VH19 2.0E−03 6 25 VH20 3.7E−04 32 25 VH201.94E+04 1.13E−03  5.84E−08 10.2 37 VH22 2.1E−03 6 25 VH23 5.7E−04 20 25VH24 6.3E−04 18 25 VH31 n.d. VH32 n.d. VH33 n.d.

Reference values VH00 with VL00 (rabbit antibody):

-   -   25° C.: kd=2.6E−04; t/2=44 minutes    -   37° C.: ka=3.7E+04, kd=5.25E−03, KD=1.4E−08, t/2=22 minutes

In the following Table the kinetic constants for different VH/VLcombinations are shown (determined according to Example 8).

KD t/2diss KD t/2diss VH/VL 25° C. 25° C. 37° C. 37° C. combination [nM][min] MR [nM] [min] MR VH00/VL00 8 54 0.6 12 24 0.8 VH20/VL22 37 16 0.468 5 0.5 VH32/VL21 18 26 0.5 32 9 0.6 VH32/VL22 14 29 0.5 31 8 0.6VH33/VL22 20 25 0.4 39 8 0.5

The biochemical binding of different combinations of humanized VH and VLis shown in FIG. 3A-FIG. 3D and FIG. 4A-FIG. 4B.

In the following Table the binding specificity for different VH/VLcombinations are shown (EC₅₀ values in [ng/ml]).

tau peptide S422A tau Tau(pS422) full length full length residues micro-mutant fragment Tau(pS422) tau 416 to 430 tubuli S422A VH/VL SEQ ID SEQID Tau(pS422) SEQ ID of SEQ ID associated SEQ ID combination NO: 03 NO:02 aggregates NO: 01 NO: 01 tau NO: 01 VH00/VL00 6.3 5.2 18.1 no >1000no 47.9 binding binding VH20/VL22 4.8 4.0 27.2 no >1000 no 110.6 bindingbinding VH32/VL21 4.4 2.9 9.4 no 634 no 21.5 binding binding VH32/VL225.6 3.5 8.3 no 48 no 17.4 binding binding VH33/VL22 5.6 3.8 13.5 no 120no 34.5 binding binding

The sensitivity of selected humanized VH/VL combinations to the humantau mutant S422A can be seen from the Western Blots shown in FIG. 5 .All humanized variants selectively bind to human tau phosphorylated atS422. There is low level cross-reactivity to non-S422 phosphoepitopes ofthe parent rabbit antibody but the humanized variants shown are lesscross-reactive in this respect than the parental rabbit antibody.

In FIG. 6 the binding to PHF-tau in brain extracts of Alzheimer'sdisease patients for the parental rabbit antibody and for selectedhumanized anti-human Tau(pS422) antibodies is shown.

Based on the above, the combination of VH32/VL22 was chosen as humanizedantibody.

The following average clearance rates in cynomolgus have been found withthe parental VH/VL combination and the humanized VH/VL combination(normal clearance for human IgG is 0.18-0.36 ml/hr/kg).

average average clearance clearance VH/VL @ 1 mg/kg @ 10 mg/kgcombination [ml/hr/kg] [ml/hr/kg] VH00/VL00 0.15 n.d. VH32/VL22 1.03 2.5n.d. = not determined

It can be seen that the VH32/VL22 combination has an increased clearancerate that is dose dependent.

In the following Table the kinetic constants for further VH/VLcombinations generated to address the increased, dose-dependentclearance rate of VH32/VL22 with Tau(pS422) fragment are shown(determined according to Example 10).

VH/VL ka kd KD combination [1/Ms] [1/s] [nM] VH35H5/VL31A1 3.80E+056.16E−04 1.62 nM VH35H5/VL35F2 1.40E+05 3.21E−04 2.29 nM VH35H5/VL49G13.12E+05 6.24E−04 2.00 nM VH76A6/VL145B12 2.40E+05 6.28E−04 2.61 nMVH00/VL00 1.35E+05 2.65E−04 1.96 nM VH32/VL35G4 1.03E+05 2.31E−04 2.25nM VH76A6/VL35G4 1.12E+05 8.65E−05 0.77 nM

In the following Table the kinetic constants for VH/VL combinations asFab fragment with full length Tau(pS422) and Tau(pS422) fragment areshown (determined according to Example 11 and Example 12).

Tau(pS422) fragment full length Tau(pS422) VH/VL ka kd KD ka kd KDcombination [1/Ms] [1/s] [nM] [1/Ms] [1/s] [nM] VH32/VL22 4.79E+052.76E−04 0.6 8.42E+05 4.23E−04 0.5 VH35H5/VL31A1 4.10E+05 1.43E−04 0.36.68E+05 2.26E−04 0.3 VH35H5/VL49G1 4.23E+05 6.03E−05 0.1 7.46E+059.38E−05 0.1

In the following Table the binding specificity for different VH/VLcombinations determined by ELISA are shown (selectivity forphosphorylated versus non-phosphorylated tau).

full length Tau(pS422) tau peptide Tau(pS422) fragment Tau(pS422) fulllength tau 416 to 430 of VH/VL SEQ ID NO: 02 SEQ ID NO: 03 aggregatesSEQ ID NO: 01 SEQ ID NO: 01 combination EC₅₀ [ng/ml] EC₅₀ [ng/ml] EC₅₀[ng/ml] OD@1 μg/mL OD@1 μg/mL VH00/VL00 26.2 40.0 62.9 0.04 0.54VH32/VL22 21.9 40.4 31.1 0.14 2.26 VH35H5/VL31A1 21.1 33.0 36.6 0.041.03 VH35H5/VL35F2 28.0 45.2 57.0 0.04 1.31 VH35H5/VL49G1 20.4 34.0 30.60.04 1.42 VH32/VL4G1 16.8 27.3 27.0 0.04 1.13 VH76A6/VL145B12 23.8 40.339.0 0.04 0.44 VH32/VL35G4 26.1 43.8 78.6 0.04 0.07 VH76A6/VL35G4 23.333.4 79.6 0.04 0.07

The following average clearance rates in cynomolgus have been found withdifferent VH/VL combinations (normal clearance for human IgG is0.18-0.36 ml/hr/kg; n.d.=not determined).

average average average clearance clearance clearance VH/VL @ 1 mg/kg @10 mg/kg @ 25 mg/kg combination [ml/hr/kg] [ml/hr/kg] [ml/hr/kg]VH00/VL00 0.15 n.d. n.d. VH32/VL22 1.03 2.5  n.d. VH35H5/VL31A1 0.140.19 0.24 VH35H5/VL35F2 0.62 n.d. n.d. VH35H5/VL49G1 0.63 n.d. n.d.VH76A6/VL35G4 0.10 n.d. n.d.

The following average clearance rates in mouse have been found withdifferent VH/VL combinations (normal clearance for human IgG is0.18-0.36 ml/hr/kg).

average average average clearance clearance clearance VH/VL @ 5 mg/kg @25 mg/kg @ 50 mg/kg combination [ml/hr/kg] [ml/hr/kg] [ml/hr/kg]VH00/VL00 0.17 0.21 n.d. VH32/VL22 0.27 n.d. 1.31 VH35H5/VL31A1 0.200.29 n.d. VH35H5/VL35F2 0.18 0.30 n.d. VH35H5/VL49G1 0.28 0.58 n.d.VH76A6/VL35G4 0.19 0.52 n.d.

In one embodiment of all aspects as reported herein the humanizedanti-human Tau(pS422) antibody has an average clearance rate afterintraveneous application of less than 0.6 ml/hr/kg at a dose of up to 25mg/kg. In one embodiment the average clearance rate is 0.3 ml/hr/kg orless at a dose of up to 25 mg/kg.

In one preferred aspect, the invention provides a (humanized) anti-humanTau(pS422) antibody comprising at least one, or two, or three, or four,or five, or six HVRs selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:71; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 73; and(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

In one preferred aspect, the invention provides a (humanized) anti-humanTau(pS422) antibody comprising at least one, or two, or three, or four,or five, or six HVRs selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:70; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72; and(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

In one preferred aspect, the invention provides a (humanized) anti-humanTau(pS422) antibody comprising at least one, or two, or three, or four,or five, or six HVRs selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

In one preferred aspect, the invention provides a (humanized) anti-humanTau(pS422) antibody comprising at least one, or two, or three, or four,or five, or six HVRs selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 77; (c) HVR-H3 comprising the amino acid sequence of SEQID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75.

In one aspect, the invention provides a (humanized) anti-humanTau(pS422) antibody comprising at least one, or two, or three, or four,or five, or six HVRs selected from (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79.

In one aspect, the invention provides a (humanized) antibody comprisingat least one, at least two, or all three VH HVR sequences selected from(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 08; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 09; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10.

In one embodiment, the antibody comprises (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO: 08; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 09; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 10.

In another embodiment the antibody further comprises at least one, atleast two, or all three VL HVR sequences selected from

-   i) (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:    71; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 73;    and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15;    or-   ii) (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:    70; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72;    and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15;    or-   iii) (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:    12; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14;    and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79.

In a further embodiment, the antibody comprises

-   i) (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:    71; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 73;    and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15;    or-   ii) (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:    70; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72;    and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15;    or-   iii) (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:    12; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14;    and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79.

In one aspect, the invention provides a (humanized) antibody comprising

-   i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:    08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:    09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:    10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:    71; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 73;    and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15,    or-   ii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:    08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:    09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:    10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:    70; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72;    and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15,    or-   iii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:    08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:    09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:    10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:    12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14;    and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79.

In another embodiment the VH or VL contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-human Tau(pS422) antibody comprisingthat sequence retains the ability to bind to human Tau(pS422).

In a further aspect of the invention, an anti-human Tau(pS422) antibodyaccording to any of the above embodiments is a monoclonal antibody,including a chimeric, humanized or human antibody. In one embodiment, ananti-human Tau(pS422) antibody is an antibody fragment, e.g., a Fv, Fab,Fab′, scFv, diabody, or F(ab′)2 fragment. In another embodiment, theantibody is a full length antibody, e.g., an intact IgG1 or IgG 4antibody or other antibody class or isotype as defined herein.

The (humanized) antibody as reported herein reduces Tau(pS422) levels inthe brain of transgenic TauPS2APP mice.

In a further aspect, a (humanized) anti-human Tau(pS422) antibodyaccording to any of the above embodiments may incorporate any of thefeatures, singly or in combination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (KD) of ≤100 nM, ≤50 nM, or between 1 nM and 100 nM (e.g.,10⁻⁷M or less, e.g., from 10⁻⁷M to 10⁻⁹M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen, Y. et al., J.Mol. Biol. 293 (1999) 865-881). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM (¹²⁵I)-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta, L. G. et al., Cancer Res. 57 (1997) 4593-4599). TheFab of interest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN) 20®) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using a BIACORE® surfaceplasmon resonance assay. For example, an assay using a BIACORE®-2000 ora BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) is performed at 25° C.with immobilized antigen CMS chips at ˜10 response units (RU). In oneembodiment, carboxymethylated dextran biosensor chips (CMS, BIAcore,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN20®) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/minute. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE ° Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on) (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999)865-881). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson, P. J. etal., Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see,e.g., Plueckthun, A., In; The Pharmacology of Monoclonal Antibodies,Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994),pp. 269-315; see also WO 93/16185; U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)2 fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 0 404 097; WO1993/01161; Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134; andHolliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448.Triabodies and tetrabodies are also described in Hudson, P. J. et al.,Nat. Med. 9 (20039 129-134).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein.

3. Humanized Antibodies

Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of or a full length human constantregion. In some embodiments, some FR residues in a humanized antibodyare substituted with corresponding residues from a non-human antibody(e.g., the antibody from which the HVR residues are derived), e.g., torestore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro, J. C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, andare further described, e.g., in Riechmann, I. et al., Nature 332 (1988)323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989)10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri, S. V. et al., Methods 36 (2005) 25-34 (describingspecificity determining region (SDR) grafting); Padlan, E. A., Mol.Immunol. 28 (1991) 489-498 (describing “resurfacing”); Dall'Acqua, W. F.et al., Methods 36 (2005) 43-60 (describing “FR shuffling”); andOsbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br.J. Cancer 83 (2000) 252-260 (describing the “guided selection” approachto FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims, M. J. et al., J. Immunol. 151 (1993) 2296-2308;framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of light or heavy chain variableregions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89(1992) 4285-4289; and Presta, L. G. et al., J. Immunol. 151 (1993)2623-2632); human mature (somatically mutated) framework regions orhuman germline framework regions (see, e.g., Almagro, J. C. andFransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regionsderived from screening FR libraries (see, e.g., Baca, M. et al., J.Biol. Chem. 272 (1997) 10678-10684 and Rosok, M. J. et al., J. Biol.Chem. 271 (19969 22611-22618).

4. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g., a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for human Tau(pS422) and the other is for any otherantigen. In certain embodiments, bispecific antibodies may bind to twodifferent epitopes of human Tau(pS422). Bispecific antibodies can beprepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein, C.and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, andTraunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole”engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specificantibodies may also be made by engineering electrostatic steeringeffects for making antibody Fc-heterodimeric molecules (WO 2009/089004);cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); usingleucine zippers to produce bi-specific antibodies (see, e.g., Kostelny,S. A. et al., J. Immunol. 148 (1992) 1547-1553; using “diabody”technology for making bispecific antibody fragments (see, e.g.,Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448);and using single-chain Fv (sFv) dimers (see, e.g., Gruber, M et al., J.Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies asdescribed, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g., US 2006/0025576).

The antibody or fragment herein also includes a “Dual Acting Fab” or“DAF” comprising an antigen binding site that binds to human Tau(pS422)as well as another, different antigen (see, US 2008/0069820, forexample).

The antibody or fragment herein also includes multispecific antibodiesdescribed in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO2010/145792, and WO 2010/145793.

5. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs.

Conservative substitutions are shown in the following Table under theheading of “preferred substitutions”. More substantial changes areprovided in the following Table under the heading of “exemplarysubstitutions”, and as further described below in reference to aminoacid side chain classes. Amino acid substitutions may be introduced intoan antibody of interest and the products screened for a desiredactivity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE Original Exemplary Conservative Residue SubstitutionsSubstitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; AlaSer Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H)Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Leu NorleucineLeu (L) Norleucine; Ile; Val; Met; Ile Ala; Phe Lys (K) Arg; Gln; AsnArg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr TyrPro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; PheTyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; LeuNorleucine

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanizedantibody). Generally, the resulting variant(s) selected for furtherstudy will have modifications (e.g., improvements) in certain biologicalproperties (e.g., increased affinity, reduced immunogenicity) relativeto the parent antibody and/or will have substantially retained certainbiological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g., bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, P. S.,Methods Mol. Biol. 207 (2008) 179-196), and/or residues that contactantigen, with the resulting variant VH or VL being tested for bindingaffinity. Affinity maturation by constructing and reselecting fromsecondary libraries has been described, e.g., in Hoogenboom, H. R. etal. in Methods in Molecular Biology 178 (2002) 1-37. In some embodimentsof affinity maturation, diversity is introduced into the variable geneschosen for maturation by any of a variety of methods (e.g., error-pronePCR, chain shuffling, or oligonucleotide-directed mutagenesis). Asecondary library is then created. The library is then screened toidentify any antibody variants with the desired affinity. Another methodto introduce diversity involves HVR-directed approaches, in whichseveral HVR residues (e.g., 4-6 residues at a time) are randomized. HVRresidues involved in antigen binding may be specifically identified,e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham, B. C. and Wells, J. A., Science244 (1989) 1081-1085. In this method, a residue or group of targetresidues (e.g., charged residues such as arg, asp, his, lys, and glu)are identified and replaced by a neutral or negatively charged aminoacid (e.g., alanine or polyalanine) to determine whether the interactionof the antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc-region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of theFc-region (see, e.g., Wright, A. and Morrison, S. L., TIBTECH 15 (1997)26-32). The oligosaccharide may include various carbohydrates, e.g.,mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, aswell as a fucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

c) Fc-region variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc-region of an antibody provided herein, therebygenerating an Fc-region variant. The Fc-region variant may comprise ahuman Fc-region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4Fc-region) comprising an amino acid modification (e.g., a substitution)at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch, J. V. and Kinet, J. P., Annu. Rev. Immunol. 9(1991) 457-492. Non-limiting examples of in vitro assays to assess ADCCactivity of a molecule of interest is described in U.S. Pat. No.5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA83 (1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad. Sci.USA 82 (1985) 1499-1502); U.S. Pat. No. 5,821,337 (see Bruggemann, M. etal., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactiveassays methods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, Calif.); and CytoTox 96® non-radioactive cytotoxicity assay(Promega, Madison, Wis.)). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998)652-656. C1q binding assays may also be carried out to confirm that theantibody is unable to bind C1q and hence lacks CDC activity. See, e.g.,C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. Toassess complement activation, a CDC assay may be performed (see, forexample, Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996)163-171; Cragg, M. S. et al., Blood 101 (2003) 1045-1052; and Cragg, M.S. and M. J. Glennie, Blood 103 (2004) 2738-2743). FcRn binding and invivo clearance/half-life determinations can also be performed usingmethods known in the art (see, e.g., Petkova, S. B. et al., Int.Immunol. 18 (2006: 1759-1769).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc-region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc-region mutantsinclude Fc-region mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc-region mutant with substitution of residues 265 and 297 toalanine (U.S. Pat. No. 7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields, R. L. et al., J. Biol. Chem. 276 (2001) 6591-6604).

In some embodiments, alterations are made in the Fc-region that resultin altered (i.e., diminished) C1q binding and/or Complement DependentCytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO99/51642, and Idusogie, E. E. et al., J. Immunol. 164 (2000) 4178-4184.

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc-regionwith one or more substitutions therein which improve binding of theFc-region to FcRn. Such Fc-region variants include those withsubstitutions at one or more of Fc-region residues: 238, 256, 265, 272,286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380,382, 413, 424 or 434, e.g., substitution of Fc-region residue 434 (U.S.Pat. No. 7,371,826).

See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc-region variants.

d) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc-region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional non-proteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and non-proteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the non-proteinaceous moiety is a carbonnanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005)11600-11605). The radiation may be of any wavelength, and includes, butis not limited to, wavelengths that do not harm ordinary cells, butwhich heat the non-proteinaceous moiety to a temperature at which cellsproximal to the antibody-non-proteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-human Tau(pS422) antibodydescribed herein is provided. Such nucleic acid may encode an amino acidsequence comprising the VL and/or an amino acid sequence comprising theVH of the antibody (e.g., the light and/or heavy chains of theantibody). In a further embodiment, one or more vectors (e.g.,expression vectors) comprising such nucleic acid are provided. In afurther embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, e.g., a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In oneembodiment, a method of making an anti-human Tau(pS422) antibody isprovided, wherein the method comprises culturing a host cell comprisinga nucleic acid encoding the antibody, as provided above, underconditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production of an anti-human Tau(pS422) antibody, nucleicacid encoding an antibody, e.g., as described above, is isolated andinserted into one or more vectors for further cloning and/or expressionin a host cell. Such nucleic acid may be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K. A., In:Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), HumanaPress, Totowa, N.J. (2003), pp. 245-254, describing expression ofantibody fragments in E. coli). After expression, the antibody may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; andLi, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES' technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TM cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

C. Assays

Anti-human Tau(pS422) antibodies provided herein may be identified,screened for, or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

1. Binding assays and other assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, alphaLISA,Western blot, antibody or reverse phase array, etc.

In an exemplary ELISA or alphaLISA assay, Tau(pS422) in solution (e.g.,in cell supernatant, cell or tissue lysates, body fluids, etc.) is boundby a capture antibody, which specifically binds to a first epitope onTau(pS422), or Tau(pS422) in a certain conformation and a detectionantibody coupled to a detection entity, which specifically binds to asecond epitope or conformation of Tau(pS422). The readout is based onthe detection entity (chemiluminescence, fluorescence, energy transferinduced luminescence, etc.). In some instances the same antibody can beused in the same assay as capture and detection antibody to detectaggregated forms of Tau(pS422) (see e.g., Tokuda, T. et al., Neurology75 (2010) 1766-1772).

In the case of antibody array, antibodies are spotted onto glass ornitrocellulose chips. The slides are blocked and incubated withTau(pS422) containing solution, washed to remove unbound antibodies andbound antibodies are detected with a fluorescently labeled correspondingsecondary antibody. The fluorescence signal is measured by afluorescence slide scanner. Similarly for a reverse phase array,recombinant Tau(pS422), cell supernatant, cell or tissue lysates, bodyfluids, etc. are spotted onto glass or nitrocellulose chips. The slidesare blocked and individual arrays are incubated with an antibody againsta specific epitope on Tau(pS422). Unbound antibodies are washed off andbound antibodies are detected with a fluorescently labeled correspondingsecondary antibody. The fluorescence signal is measured by afluorescence slide scanner (Dernick, G., et al., J. Lipid Res. 52 (2011)2323-2331).

In the example of Western blot, aggregated recombinant Tau(pS422) orTau(pS422) derived, e.g., from cell supernatant, cell or tissue lysates,body fluids, etc. is separated by molecular weight in SDS PAGE or nativegel conditions and blotted onto a nitrocellulose or PVDF membrane. Afterblocking the membrane is incubated with antibodies specific to aminoacid sequence or conformations of Tau(pS422). Thereafter the membrane iswashed to remove unbound antibody. Bound antibodies are detected bycorresponding secondary antibodies coupled to detection entities forchemiluminescence or fluorescence or other means of detection.Antibodies specific to amino acid sequences of Tau(pS422) will bind toTau(pS422) in various aggregated forms and hence molecular weights aslong as the epitope is not masked by the aggregation. On the other hand,conformation specific antibodies will detect only certain aggregatedforms of Tau(pS422) revealing only bands at specific molecular weights(see, e.g., Towbin, H., et al., Proc. Natl. Acad. Sci. USA 76 (1979)4350-4353; Burnette, W. N., Anal. Biochem. 112 (1981) 195-203).

In another aspect, competition assays may be used to identify anantibody that competes with the (humanized) antibody as reported hereinfor binding to human Tau(pS422). In certain embodiments, such acompeting antibody binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by the (humanized) antibody asreported herein. Detailed exemplary methods for mapping an epitope towhich an antibody binds are provided in Morris, G. E. (ed.), EpitopeMapping Protocols, In: Methods in Molecular Biology, Vol. 66, HumanaPress, Totowa, N.J. (1996).

In an exemplary competition assay, immobilized human Tau(pS422) isincubated in a solution comprising a first labeled antibody that bindsto human Tau(pS422) and a second unlabeled antibody that is being testedfor its ability to compete with the first antibody for binding to humanTau(pS422). As a control, immobilized human Tau(pS422) is incubated in asolution comprising the first labeled antibody but not the secondunlabeled antibody. After incubation under conditions permissive forbinding of the first antibody to human Tau(pS422), excess unboundantibody is removed, and the amount of label associated with immobilizedhuman Tau(pS422) is measured. If the amount of label associated withimmobilized human Tau(pS422) is substantially reduced in the test samplerelative to the control sample, then that indicates that the secondantibody is competing with the first antibody for binding to humanTau(pS422) (see, e.g., Harlow, E. and Lane, D., Antibodies: A LaboratoryManual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1988)).

2. Activity assays

In one aspect, assays are provided for identifying anti-human Tau(pS422)antibodies thereof having biological activity. Biological activity mayinclude, e.g., protection from/reduction of/inhibition ofTau(pS422)-induced cytotoxicity, and/or protection from/reductionof/inhibition of cell-to-cell transmission of oligomeric humanTau(pS422), and/or reduction of Tau(pS422)-induced caspase activity inLUHMES cells. Antibodies having such biological activity in vivo and/orin vitro are also provided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity.

The protective biological activity can be assessed by adding conditionedmedium containing secreted Tau(pS422), which causes cell death onrecipient neuronal cells. This toxicity can be reversed by addingprotective antibodies as described herein. The toxic nature of secretedTau(pS422) has been established previously (Emmanouilidou, E., et al.,J. Neurosci., 30 (2010) 6838-6851).

D. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-human Tau(pS422) antibodiesprovided herein are useful for detecting the presence of humanTau(pS422) in a biological sample. The term “detecting” as used hereinencompasses quantitative or qualitative detection. In certainembodiments, a biological sample comprises a cell or tissue, such asbrain tissue.

In one embodiment, an anti-human Tau(pS422) antibody for use in a methodof diagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of human Tau(pS422) in a biological sample isprovided. In certain embodiments, the method comprises contacting thebiological sample with an anti-human Tau(pS422) antibody as describedherein under conditions permissive for binding of the anti-humanTau(pS422) antibody to human Tau(pS422), and detecting whether a complexis formed between the anti-human Tau(pS422) antibody and humanTau(pS422). Such method may be an in vitro or in vivo method. In oneembodiment, an anti-human Tau(pS422) antibody is used to select subjectseligible for therapy with an anti-human Tau(pS422) antibody, e.g., wherehuman Tau(pS422) is a biomarker for selection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include neurodegeneration with brain iron accumulation type 1(NBIA1), pure autonomic failure, Down's syndrome, complex of Guam, andseveral Lewy body disorders, such as diffuse Lewy body disease (DLBD),the Lewy body variant of Alzheimer's disease (LBVAD), certain forms ofGaucher's disease, and Parkinson's disease dementia (PDD).

In certain embodiments, labeled anti-human Tau(pS422) antibodies areprovided. Labels include, but are not limited to, labels or moietiesthat are detected directly (such as fluorescent, chromophoric,electron-dense, chemiluminescent, and radioactive labels), as well asmoieties, such as enzymes or ligands, that are detected indirectly,e.g., through an enzymatic reaction or molecular interaction. Exemplarylabels include, but are not limited to, the radioisotopes ³²P, ¹⁴C,¹²⁵I, ³H, and ¹³¹I, fluorophores such as rare earth chelates orfluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, luceriferases, e.g., firefly luciferase and bacterialluciferase (U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkalinephosphatase, β-galactosidase, glucoamylase, lysozyme, saccharideoxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

E. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-human Tau(pS422) antibody asdescribed herein are prepared by mixing such antibody having the desireddegree of purity with one or more optional pharmaceutically acceptablecarriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.(ed.) (1980)), in the form of lyophilized formulations or aqueoussolutions. Pharmaceutically acceptable carriers are generally nontoxicto recipients at the dosages and concentrations employed, and include,but are not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyl dimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rhuPH20, are described in US 2005/0260186 and US2006/0104968. In one aspect, a sHASEGP is combined with one or moreadditional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, where the indication being treated is Alzheimer'sdisease or prodromal Alzheimer's disease, the pharmaceutical formulationmay also contain one or more additional active ingredients such asdonepezil, memantine, rivastigmine, galantamine, rivastigmine, ergoloidmesylates, an anti-Abeta antibody and an anti-alpha-synuclein antibody.Such active ingredients are suitably present in combination in amountsthat are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methyl methacrylate) microcapsules, respectively, in colloidaldrug delivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed.) (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

F. Therapeutic Methods and Compositions

Any of the anti-human Tau(pS422) antibodies provided herein may be usedin therapeutic methods.

In one aspect, an anti-human Tau(pS422) antibody for use as a medicamentis provided. In further aspects, an anti-human Tau(pS422) antibody foruse in treating Alzheimer's disease is provided. In certain embodiments,an anti-human Tau(pS422) antibody for use in a method of treatment isprovided. In certain embodiments, the invention provides an anti-humanTau(pS422) antibody for use in a method of treating an individual havingAlzheimer's disease comprising administering to the individual aneffective amount of the anti-human Tau(pS422) antibody. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent, e.g.,as described above. In further embodiments, the invention provides ananti-human Tau(pS422) antibody for use in inhibiting Tau(pS422)-inducedcytotoxicity in human neurons and glia cells, or inhibiting cell-to-celltransmission of oligomeric human Tau(pS422) between neurons and gliacells, or reducing Tau(pS422)-induced caspase activity. In certainembodiments, the invention provides an anti-human Tau(pS422) antibodyfor use in a method of inhibiting Tau(pS422)-induced cytotoxicity inhuman neurons and glia cells, or inhibiting cell-to-cell transmission ofoligomeric human Tau(pS422) between neurons and glia cells, or reducingTau(pS422)-induced caspase activity in an individual comprisingadministering to the individual an effective of the anti-humanTau(pS422) antibody to inhibit Tau(pS422)-induced cytotoxicity in humanneurons and glia cells, or inhibit cell-to-cell transmission ofoligomeric human Tau(pS422) between neurons and glia cells, or reduceTau(pS422)-induced caspase activity. An “individual” according to any ofthe above embodiments is preferably a human.

In a further aspect, the invention provides for the use of an anti-humanTau(pS422) antibody in the manufacture or preparation of a medicament.In one embodiment, the medicament is for treatment of Alzheimer'sdisease. In a further embodiment, the medicament is for use in a methodof treating Alzheimer's disease comprising administering to anindividual having Alzheimer's disease an effective amount of themedicament. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent. In a further embodiment, the medicament isfor inhibiting Tau(pS422)-induced cytotoxicity in human neurons and gliacells, or for inhibiting cell-to-cell transmission of oligomeric humanTau(pS422) between neurons and glia cells, or for reducingTau(pS422)-induced caspase activity. In a further embodiment, themedicament is for use in a method of inhibiting Tau(pS422)-inducedcytotoxicity in human neurons and glia cells, or inhibiting cell-to-celltransmission of oligomeric human Tau(pS422) between neurons and gliacells, or reducing Tau(pS422)-induced caspase activity in an individualcomprising administering to the individual an amount effective of themedicament to inhibit Tau(pS422)-induced cytotoxicity in human neuronsand glia cells, or to inhibit cell-to-cell transmission of oligomerichuman Tau(pS422) between neurons and glia cells, or to reduceTau(pS422)-induced caspase activity. An “individual” according to any ofthe above embodiments may be a human.

In a further aspect, the invention provides a method for treatingAlzheimer's disease. In one embodiment, the method comprisesadministering to an individual having such Alzheimer's disease aneffective amount of an anti-human Tau(pS422) antibody. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent. An“individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for inhibitingTau(pS422)-induced cytotoxicity in human neurons and glia cells, orinhibiting cell-to-cell transmission of oligomeric human Tau(pS422)between neurons and glia cells, or reducing Tau(pS422)-induced caspasein an individual. In one embodiment, the method comprises administeringto the individual an effective amount of an anti-human Tau(pS422)antibody to inhibit Tau(pS422)-induced cytotoxicity in human neurons andglia cells, or to inhibit cell-to-cell transmission of oligomeric humanTau(pS422) between neurons and glia cells, or to reduceTau(pS422)-induced caspase activity. In one embodiment, an “individual”is a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-human Tau(pS422) antibodies provided herein,e.g., for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical formulation comprises any of the anti-humanTau(pS422) antibodies provided herein and a pharmaceutically acceptablecarrier. In another embodiment, a pharmaceutical formulation comprisesany of the anti-human Tau(pS422) antibodies provided herein and at leastone additional therapeutic agent.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent or agents. In one embodiment, administration of theanti-human Tau(pS422) antibody and administration of an additionaltherapeutic agent occur within about one month, or within about one, twoor three weeks, or within about one, two, three, four, five, or sixdays, of each other.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.,by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.5 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.,every week or every three weeks (e.g., such that the patient receivesfrom about two to about twenty, or e.g., about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-human Tau(pS422) antibody.

III. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-human Tau(pS422) antibody.

IV. Specific Embodiments

-   1. A humanized antibody that specifically binds to human Tau(pS422),    wherein the antibody comprises    -   a) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        09 and 10, or b) in the heavy chain variable domain the HVRs of        SEQ ID NO: 08, 77 and 10.-   2. The humanized antibody according to item 1, further comprising    -   a) in the light chain variable domain the HVRs of SEQ ID NO: 71,        73 and 15, or b) in the light chain variable domain the HVRs of        SEQ ID NO: 70, 72 and 15, or c) in the light chain variable        domain the HVRs of SEQ ID NO: 12, 14 and 79, or d) in the light        chain variable domain the HVRs of SEQ ID NO: 71, 81 and 15,        or e) in the light chain variable domain the HVRs of SEQ ID NO:        12, 14 and 74, or f) in the light chain variable domain the HVRs        of SEQ ID NO: 12, 14 and 75.-   3. The humanized antibody according to any one of items 1 to 2,    comprising    -   a) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        09 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 71, 73 and 15, or    -   b) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        09 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 70, 72 and 15, or    -   c) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        77 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 12, 14 and 79, or    -   d) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        09 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 12, 14 and 79, or    -   e) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        77 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 71, 81 and 15, or    -   f) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        09 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 12, 14 and 74, or    -   g) in the heavy chain variable domain the HVRs of SEQ ID NO: 08,        77 and 10, and in the light chain variable domain the HVRs of        SEQ ID NO: 12, 14 and 75.-   4. The humanized antibody according to any one of items 1 to 3,    comprising    -   a) a heavy chain variable domain of SEQ ID NO: 19 and a light        chain variable domain of SEQ ID NO: 67, or    -   b) a heavy chain variable domain of SEQ ID NO: 65 and a light        chain variable domain of SEQ ID NO: 66, or    -   c) a heavy chain variable domain of SEQ ID NO: 76 and a light        chain variable domain of SEQ ID NO: 78, or    -   d) a heavy chain variable domain of SEQ ID NO: 19 and a light        chain variable domain of SEQ ID NO: 78, or    -   e) a heavy chain variable domain of SEQ ID NO: 76 and a light        chain variable domain of SEQ ID NO: 80, or    -   f) a heavy chain variable domain of SEQ ID NO: 65 and a light        chain variable domain of SEQ ID NO: 67, or    -   g) a heavy chain variable domain of SEQ ID NO: 65 and a light        chain variable domain of SEQ ID NO: 68, or    -   h) a heavy chain variable domain of SEQ ID NO: 76 and a light        chain variable domain of SEQ ID NO: 69.-   5. A humanized bispecific antibody comprising    -   i) a first binding site selected from    -   a) a heavy chain variable domain of SEQ ID NO: 20 and a light        chain variable domain of SEQ ID NO: 17, or    -   b) a heavy chain variable domain of SEQ ID NO: 19 and a light        chain variable domain of SEQ ID NO: 16, or    -   c) a heavy chain variable domain of SEQ ID NO: 19 and a light        chain variable domain of SEQ ID NO: 17, or    -   d) a heavy chain variable domain of SEQ ID NO: 21 and a light        chain variable domain of SEQ ID NO: 17, or    -   e) a heavy chain variable domain of SEQ ID NO: 65 and a light        chain variable domain of SEQ ID NO: 67, or    -   f) a heavy chain variable domain of SEQ ID NO: 19 and a light        chain variable domain of SEQ ID NO: 66, or    -   g) a heavy chain variable domain of SEQ ID NO: 76 and a light        chain variable domain of SEQ ID NO: 78, or    -   h) a heavy chain variable domain of SEQ ID NO: 65 and a light        chain variable domain of SEQ ID NO: 78, or    -   i) a heavy chain variable domain of SEQ ID NO: 65 and a light        chain variable domain of SEQ ID NO: 66, or    -   j) a heavy chain variable domain of SEQ ID NO: 65 and a light        chain variable domain of SEQ ID NO: 68, or    -   k) a heavy chain variable domain of SEQ ID NO: 76 and a light        chain variable domain of SEQ ID NO: 69, or    -   1) a heavy chain variable domain of SEQ ID NO: 76 and a light        chain variable domain of SEQ ID NO: 80,    -   and    -   ii) a second binding site selected from    -   a) a heavy chain variable domain of SEQ ID NO: 82 and a light        chain variable domain of SEQ ID NO: 85, or    -   b) a heavy chain variable domain of SEQ ID NO: 83 and a light        chain variable domain of SEQ ID NO: 85, or    -   c) a heavy chain variable domain of SEQ ID NO: 84 and a light        chain variable domain of SEQ ID NO: 85.-   6. The humanized antibody according to any one of items 1 to 5,    wherein the antibody is for use in the treatment of Alzheimer's    disease.-   7. The humanized antibody according to any one of items 1 to 6,    wherein the antibody is effector function silent.-   8. The humanized antibody according to any one of items 1 to 7,    wherein the antibody has no effector function.-   9. The humanized antibody according to any one of items 1 to 8,    wherein the antibody    -   i) specifically binds to a polypeptide that has the amino acid        sequence of SEQ ID NO: 03, and/or    -   ii) does not bind to full length human tau (SEQ ID NO: 01) at 1        μg/mL, and/or    -   iii) specifically binds to full length human tau phosphorylated        at the serine at position 422 (SEQ ID NO: 02), and/or    -   iv) specifically binds to aggregates of human tau phosphorylated        at the serine at position 422 (SEQ ID NO: 02).-   10. The humanized antibody according to any one of items 2 to 9,    wherein the antibody has an EC₅₀ value for    -   a) the human Tau(pS422) fragment that has the amino acid        sequence of SEQ ID NO: 03 of 6 ng/mL or less, and/or    -   b) the full length human Tau(pS422) that has the amino acid        sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or    -   c) aggregates of human Tau(pS422) that has the amino acid        sequence of SEQ ID NO:

02 of 30 ng/mL or less, and/or

-   -   d) the human tau that has the amino acid sequence of SEQ ID NO:        01 and that has the amino acid mutation S422A of 125 ng/mL or        less.

-   11. The humanized antibody according to any one of items 1 to 10,    wherein the antibody specifically binds to human Tau(pS422) (SEQ ID    NO: 02) and does not bind to human tau (SEQ ID NO: 01).

-   12. The humanized antibody according to any one of items 1 to 11,    wherein the antibody has in the heavy chain variable domain at    positions 4, 24 and 78, a valine residue.

-   13. The humanized antibody according to any one of items 1 to 12,    wherein the antibody has in the heavy chain variable domain at    position 71, an arginine residue.

-   14. The humanized antibody according to any one of items 1 to 13,    wherein the antibody is a monoclonal antibody.

-   15. The humanized antibody according to any one of items 1 to 14,    wherein the antibody is an antibody fragment that binds to human    Tau(pS422) and    -   i) specifically binds to a polypeptide that has the amino acid        sequence of SEQ ID NO: 03, and/or    -   ii) does not bind to full length human tau (SEQ ID NO: 01) at 1        μg/mL, and/or    -   iii) specifically binds to full length human tau phosphorylated        at the serine at position 422 (SEQ ID NO: 02), and/or    -   iv) specifically binds to aggregates of human tau phosphorylated        at the serine at position 422 (SEQ ID NO: 02), and/or    -   v) specifically binds to full length human tau that has the        amino acid sequence of SEQ ID NO: 01 and that has the amino acid        mutation S422A, and/or    -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that has        the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or less,        and/or    -   vii) has an EC₅₀ value for the full length human Tau(pS422) that        has the amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or        less, and/or    -   viii) has an EC₅₀ value for aggregates of human Tau(pS422) that        has the amino acid sequence of SEQ ID NO: 02 of 30 ng/mL or        less, and/or    -   ix) has an EC₅₀ value for the human tau that has the amino acid        sequence of SEQ ID NO: 01 and that has the amino acid mutation        S422A of 125 ng/mL or less.

-   16. The humanized antibody according to any one of items 1 to 14,    wherein the antibody is    -   a) a full length antibody of the human subclass IgG1, or    -   b) a full length antibody of the human subclass IgG4, or    -   c) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A and P329G,    -   d) a full length antibody of the human subclass IgG4 with the        mutations S228P, L235E and P329G,    -   e) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A and P329G in both heavy chains and the        mutations T366W and S354C in one heavy chain and the mutations        T366S, L368A, Y407V and Y349C in the respective other heavy        chain, or    -   f) a full length antibody of the human subclass IgG4 with the        mutations S228P and P329G in both heavy chains and the mutations        T366W and S354C in one heavy chain and the mutations T366S,        L368A, Y407V and Y349C in the respective other heavy chain.

-   17. A humanized anti-human Tau(pS422) antibody, wherein    -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 08,            SEQ ID NO: 09 and SEQ ID NO: 10,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue can be present or            absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 71,            SEQ ID NO: 73 and SEQ ID NO: 15,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

-   18. A humanized anti-human Tau(pS422) antibody, wherein    -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 08,            SEQ ID NO: 09 and SEQ ID NO: 10,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue can be present or            absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 70,            SEQ ID NO: 72 and SEQ ID NO: 15,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

-   19. A humanized anti-human Tau(pS422) antibody, wherein    -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 08,            SEQ ID NO: 09 and SEQ ID NO: 10,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue can be present or            absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 12,            SEQ ID NO: 14 and SEQ ID NO: 74,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

-   20. A humanized anti-human Tau(pS422) antibody, wherein    -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 08,            SEQ ID NO: 77 and SEQ ID NO: 10,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue can be present or            absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain comprises the HVRs of SEQ ID NO: 12,            SEQ ID NO: 14 and SEQ ID NO: 75,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

-   21. A humanized anti-human Tau(pS422) antibody, wherein    -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 65,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue can be present or            absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 67,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

-   22. A humanized anti-human Tau(pS422) antibody, wherein    -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 65,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue can be present or            absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 66,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

-   23. A humanized anti-human Tau(pS422) antibody, wherein    -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 65,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue can be present or            absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 68,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

-   24. A humanized anti-human Tau(pS422) antibody, wherein    -   a) the antibody comprises two antibody heavy chains each        comprising a heavy chain variable domain and a heavy chain        constant region, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 76,        -   ii) the constant region is a human IgG1 constant region,            wherein the C-terminal lysine residue can be present or            absent, and        -   iii) the constant region comprises the amino acid changes            L234A, L235A and P329G,    -   b) the antibody comprises two antibody light chains each        comprising a light chain variable domain and a light chain        constant domain, wherein        -   i) the variable domain has the amino acid sequence of SEQ ID            NO: 69,        -   ii) the constant region is a human kappa light chain            constant region or a human lambda light chain constant            region,    -   and    -   c) the antibody        -   i) specifically binds to a polypeptide that has the amino            acid sequence of SEQ ID NO: 03, and/or        -   ii) does not bind to full length human tau (SEQ ID NO: 01)            at 1 μg/mL, and/or        -   iii) specifically binds to full length human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   iv) specifically binds to aggregates of human tau            phosphorylated at the serine at position 422 (SEQ ID NO:            02), and/or        -   v) specifically binds to full length human tau that has the            amino acid sequence of SEQ ID NO: 01 and that has the amino            acid mutation S422A, and/or        -   vi) has an EC₅₀ value for the human Tau(pS422) fragment that            has the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or            less, and/or        -   vii) has an EC₅₀ value for the full length human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 4.5            ng/mL or less, and/or        -   viii) has an EC₅₀ value for aggregates of human Tau(pS422)            that has the amino acid sequence of SEQ ID NO: 02 of 30            ng/mL or less, and/or        -   ix) has an EC₅₀ value for the human tau that has the amino            acid sequence of SEQ ID NO: 01 and that has the amino acid            mutation S422A of 125 ng/mL or less.

-   25. The humanized antibody according to any one of items 1 to 24,    wherein the antibody has in the light chain variable domain at    position 32, a lysine (K) amino acid residue (numbering according to    Kabat).

-   26. An isolated nucleic acid encoding a humanized antibody according    to any one of items 1 to 25.

-   27. A host cell comprising the nucleic acid according to item 26.

-   28. A method of producing a humanized antibody according to any one    of items 1 to 25 comprising the steps of culturing the host cell as    reported herein so that the humanized antibody is produced.

-   29. The method according to item 28, further comprising the step of    recovering the humanized antibody from the cell or the cultivation    medium.

-   30. A pharmaceutical formulation comprising the humanized antibody    according to any one of items 1 to 25 and a pharmaceutically    acceptable carrier.

-   31. The pharmaceutical formulation according to item 30, further    comprising an additional therapeutic agent.

-   32. The pharmaceutical formulation according to item 31, wherein the    additional therapeutic agent is an anti-amyloid therapeutic agent.

-   33. The pharmaceutical formulation according to item 32, wherein the    anti-amyloid therapeutic agent is an anti-human alpha-synuclein    antibody or an anti-Abeta antibody.

-   34. The humanized antibody according to any one of items 1 to 25 for    use as a medicament.

-   35. The humanized antibody according to any one of items 1 to 25 for    use in treating Alzheimer's disease.

-   36. The humanized antibody according to any one of items 1 to 25 for    use in treating prodromal Alzheimer's disease.

-   37. The humanized antibody according to any one of items 1 to 25 for    use in treating mild Alzheimer's disease.

-   38. The humanized antibody according to any one of items 1 to 25 for    use in reducing Tau(pS422)-induced neurodegeneration.

-   39. The humanized antibody according to any one of items 1 to 25 for    use in maintaining cognition and function.

-   40. The humanized antibody according to any one of items 1 to 25 for    use in slowing the rate of cognitive and functional decline.

-   41. Use of the humanized antibody according to any one of items 1 to    25 in the manufacture of a medicament.

-   42. Use according to any one of items 34 and 41, wherein the    medicament is for treatment of Alzheimer's disease.

-   43. Use according to any one of items 34 and 41 to 42, wherein the    medicament is for treatment of prodromal Alzheimer's disease.

-   44. Use according to any one of items 34 and 41 to 43, wherein the    medicament is for treatment of mild Alzheimer's disease.

-   45. Use according to any one of items 34 and 41 to 44, wherein the    medicament is for reducing Tau(pS422)-induced neurodegeneration.

-   46. Use according to any one of items 34 and 41 to 45, wherein the    medicament is for maintaining cognition and function.

-   47. Use according to any one of items 34 and 41 to 46, wherein the    medicament is for slowing the rate of cognitive and functional    decline.

-   48. A method of treating an individual having Alzheimer's disease    comprising administering to the individual an effective amount of    the humanized anti-human Tau(pS422) antibody according to any one of    items 1 to 25.

-   49. A method of reducing Tau(pS422)-induced neurodegeneration in an    individual comprising administering to the individual an effective    amount of the humanized anti-human Tau(pS422) antibody according to    any one of items 1 to 25 to reduce Tau(pS422)-induced    neurodegeneration.

-   50. A method of maintaining cognition and function in an individual    comprising administering to the individual an effective amount of    the humanized anti-human Tau(pS422) antibody according to any one of    items 1 to 25 to maintain cognition and function.

-   51. A method of slowing the rate of cognitive and functional decline    in an individual comprising administering to the individual an    effective amount of the humanized anti-human Tau(pS422) antibody    according to any one of items 1 to 25 to slow the rate of cognitive    and functional decline.

-   52. Use of the humanized anti-human Tau(pS422) antibody according to    any one of items 1 to 25 in the reduction of Tau(pS422)-induced    neurodegeneration.

-   53. Use of the humanized anti-human Tau(pS422) antibody according to    any one of items 1 to 25 in maintaining cognition and function.

-   54. Use of the humanized anti-human Tau(pS422) antibody according to    any one of items 1 to 25 in slowing the rate of cognitive and    functional decline.

-   55. The humanized antibody according to any one of items 1 to 25,    wherein the antibody i) binds to Tau(pS422) on brain sections of    Tau(pS422) transgenic mice and Alzheimer's disease patients; and/or    labels Tau(pS422) in Tau(pS422) transgenic cells.

-   56. The humanized antibody according to any one of items 1 to 25,    wherein the antibody specifically binds to/recognizes early and late    stage disease-relevant forms of human Tau(p S422).

-   57. The use of the humanized antibody according to any one of items    1 to 25 for the prevention of human Tau (pS422)-related Alzheimer's    disease spread.

-   58. The use of the humanized antibody according to any one of items    1 to 25 for the reduction of lysosomal membrane disintegration.

-   59. The use of the humanized antibody according to any one of items    1 to 25 for the stabilization of lysosome membrane against human    Tau(pS422)-induced destabilization and/or disintegration.

-   60. The use of the humanized antibody according to any one of items    1 to 25 for the prevention of Alzheimer's disease progression.

V. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Materials and Methods

Recombinant DNA techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene and Oligonucleotide Synthesis

Desired gene segments were prepared by chemical synthesis at GeneartGmbH (Regensburg, Germany). The synthesized gene fragments were clonedinto an E. coli plasmid for propagation/amplification. The DNA sequencesof subcloned gene fragments were verified by DNA sequencing.Alternatively, short synthetic DNA fragments were assembled by annealingchemically synthesized oligonucleotides or via PCR. The respectiveoligonucleotides were prepared by metabion GmbH (Planegg-Martinsried,Germany).

Reagents

All commercial chemicals, antibodies and kits were used as providedaccording to the manufacturer's protocol if not stated otherwise.

Example 1

Preparation and purification of rabbit antibodies

Immunization

New Zealand White (NZW) rabbits from Charles River LaboratoriesInternational, Inc. were used for immunization. Phosphopeptide tau(416-430)[pS422] coupled on keyhole limpet hemocyanin (KLH) was solvedin K₃PO₄ puffer, pH 7.0 at a concentration of 1 mg/ml and mixed (1:1)with complete Freund's adjuvant (CFA) till generation of stabileemulsion. Three rabbits received an intra-dermal (i.d.) injection of 2ml of emulsion followed by a second intra muscular (i.m.) and thirdsubcutaneous (s.c.) injection each with 1 ml in one week interval. Thefourth i.m. injection of 1 ml was performed two weeks later followed bytwo further s.c. injections of 1 ml in four weeks interval. 10 mlperipheral whole blood samples of each animal was collected 4-6 daysafter third, fourth, fifth and sixth injection and used for single cellsorting in FACS. An additional 0.5 ml serum of each animal was collectedat the same time and used for the determination of tau (416-463)[pS422]specific antibody response.

Antibody Response

The antibody response to the immunization was determined by serialdilution of sera using an ELISA, in which 30 ng per well of biotinylatedtau (416-430)[pS422] was incubated in 1×PBS at 4° C. overnight onstreptavidin pre-coated 96-well microtiter plates (MC1347, Micro CoatBiotechnologie GmbH, Bernried, Germany). For detection, goat anti-rabbitIgG linked to a horseradish peroxidase (The Jackson laboratory) was usedat 1:16000 dilution. BM Blue POD Substrate, precipitating tetramethylbenzidine (TMB), ready-to-use solution from Roche Diagnostics GmbH wasused for visualization. Reaction was stopped via 1 N HCl and measured inTecan Infinite by 450/690 nm.

B-Cell Cloning

Coating of Plates

Sterile streptavidin-coated 6-well plates (cell culture grade) wereincubated with either a mixture of 3 biotinylated control peptides(non-phosphorylated tau (416-430), MCAK_Human (88-102)[95-pSer] andMAP2_Human (1802-1816)[pSer-1802]) or with the biotinylatedphospho-peptide tau (416-430)[pS422] each in a concentration at 0.5-1μg/ml in PBS at room temperature for 1 hour. Plates were washed insterile PBS three times before use. Cell culture 6-well plates werecoated with 2 μg/ml KLH (keyhole limpet hemocyanin) in carbonate buffer(0.1 M sodium bicarbonate, 34 mM Disodiumhydrogencarbonate, pH 9.55)overnight at 4° C. Plates were washed in sterile PBS three times beforeuse.

Isolation of rabbit peripheral blood mononuclear cells (PBMC)

EDTA containing whole blood was diluted twofold with 1×PBS beforedensity centrifugation on lympholyte mammal (Cedarlane Laboratories)which was performed to isolate rabbit PBMC. PBMCs were washed twicebefore staining with antibodies.

EL-4 B5 medium

RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented with 10% FCS(Hyclone, Logan, Utah, USA), 2 mM glutamine, 1% penicillin/streptomycinsolution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES(PAN Biotech, Aidenbach, Germany) and 0.05 mM beta-mercaptoethanol(Gibco, Paisley, Scotland).

Depletion of macrophages/monocytes

Sterile 6-well plates (cell culture grade) were used to depletemacrophages and monocytes through unspecific adhesion. Wells were eithercoated with KLH (keyhole limpet hemocyanin) or with streptavidin and thecontrol peptides. Each well was filled with at maximum 4 ml medium andup to 6×10⁶ peripheral blood mononuclear cells from the immunized rabbitand allowed to bind for 1 hour at 37° C. in the incubator. 50% of thecells in the supernatant were used for the panning step; the remaining50% of cells were directly subjected to immune fluorescence staining andsingle cell sorting.

Panning B-cells on peptides

6-well tissue culture plates coated with streptavidin and thebiotinylated peptide tau (416-430)[pS422] were seeded with up to 6×10⁶cells per 4 ml medium and allowed to bind for 1 hour at 37° C. in theincubator. Non-adherent cells were removed by carefully washing thewells 1-2 times with 1×PBS. The remaining sticky cells were detached bytrypsin for 10 minutes at 37° C. in the incubator and then washed twicein media. The cells were kept on ice until the immune fluorescencestaining.

Immune fluorescent staining and single cell sorting

Anti-rabbit IgG FITC used for single cell sorting was from AbD Serotec(STAR121F, Dusseldorf, Germany). For surface staining, cells from thedepletion and panning step were incubated with anti-rabbit IgG FITCantibody in PBS for 30 minutes rolling in the cold room at 4° C. in thedark. Following centrifugation, the supernatants were removed byaspiration. The PBMCs were subjected to 2 cycles of centrifugation andwashing with ice cold PBS. Finally the PBMCs were resuspended in icecold PBS and immediately subjected to the FACS analysis. Propidiumiodide at a concentration of 5 μg/ml (BD Pharmingen, San Diego, Calif.,USA) was added prior to the FACS analyses to discriminate between deadand live cells. FACS was performed using a Becton Dickinson FACSAriaequipped the FACSDiva software (BD Biosciences, USA) and single,FITC-labeled, live cells were deposited in 96-well plates.

B-cell culture

B-cell cultures were prepared by a method similar to that described byZubler, R. H. et al., J. Immunol. 134 (1985) 3662-3668. Briefly, singlesorted B cells were cultured in 96-well plates with 210 μl/well EL-4 B5medium with Pansorbin Cells (1:20000) (Calbiochem (Merck), Darmstadt,Deutschland), 5% rabbit thymocyte supernatant and gamma-irradiatedEL-4-B5 murine thymoma cells (2×10⁴/well) for 7 days at 37° C. in anatmosphere of 5% CO₂ in the incubator. B cell culture supernatants wereremoved for screening and the cells harvested immediately for variableregion gene cloning or frozen at −80° C. in 100 μl RLT buffer (Qiagen,Hilden, Germany).

B-cell clone screening

B-cell culture supernatants were screened for binding to biotinylatedtau (416-430)[pS422] by ELISA. Non-phosphorylated tau (416-430), KLH(keyhole limpet hemocyanin) and the unrelated phospho-peptide MCAK_Human(88-102)[95-pSer] were used as control antigens. For the preparation ofELISA plates, streptavidin pre-coated microtiter plates were incubatedwith biotinylated tau (415-430)[pS422] at 50 ng/ml for 1 hour at roomtemperature. Coating with KLH or control peptides was performed at 1 Bcell supernatants were diluted 1:5 to 1:10 and were incubated in theantigen coated microtiter plates for 60 minutes. After intensivewashing, the binding of the rabbit antibodies was detected using a sheepanti-rabbit IgG digoxigenin conjugated detection antibody (ChemiconAQ301D). After incubation with TMB at room temperature, absorbance at370 nm-492 nm was measured. B-cell clones yielding signals abovebackground with biotinylated tau (416-430)[pS422] but not with KLH andMCAK_Human (88-102)[95-pSer] were further considered and subjected tovariable region gene cloning.

PCR Amplification of V-Domains and Sequencing

Total RNA was prepared using the NucleoSpin® 8/96 RNA kit(Macherey&Nagel; 740709.4, 740698) according to manufacturer's protocol.All steps were done on an epMotion 5075 liquid handling system(Eppendorf). RNA was eluted with 60 μl RNAse free water. 6 μl of RNA wasused to generate cDNA by reverse transcriptase reaction using theSuperscript III First-Strand Synthesis SuperMix (Invitrogen 18080-400)and an oligo dT-primer according to the manufacturer's instructions. 4μl of cDNA were used to amplify the immunoglobulin heavy and light chainvariable regions (VH and VL) with the AccuPrime SuperMix (Invitrogen12344-040) in a final volume of 50 μl using the primers rbHCfinal.up andrbHCfinal.do for the heavy chain and rbLCfinal.up and rbLCfinal.do forthe light chain (see Table below). The PCR conditions were as follows:Hot start at 94° C. for 5 minutes; 35 cycles of 20 seconds at 94° C., 20seconds at 70° C., 45 seconds at 68° C., and a final extension at 68° C.for 7 minutes.

TABLE rbHCfinal.up AAGCTTGCCACCATGGAGAC (SEQ ID NO: 61)TGGGCTGCGCTGGCTTC rbHCfinal.do CCATTGGTGAGGGTGCCCGAG (SEQ ID NO: 62)rbLCfinal.up AAGCTTGCCACCATGGACAYGA (SEQ ID NO: 63) GGGCCCCCACTCrbLCfinal.do CAGAGTRCTGCTGAGGTTGTAGG (SEQ ID NO: 64) TAC

8 μl of the 50 μl PCR solution were loaded on a 48 E-Gel 2% (InvitrogenG8008-02). Positive PCR reactions were cleaned using the NucleoSpin®Extract II kit (Macherey&Nagel; 740609250) according to manufacturer'sprotocol and eluted in 50 μl elution buffer. 12 μl of purified PCRproducts were sequenced directly in both directions using therbHCfinal.up and rbHCfinal.do for heavy chains and rbLCfinal.up andrbLCfinal.do for light chains (see Table above).

Recombinant expression of rabbit monoclonal antibodies and rabbit/mousechimeric antibodies

For recombinant expression of rabbit monoclonal antibodies, PCR-productscoding for VH or VL were cloned as cDNA into expression vectors by theoverhang cloning method (Haun, R. S. et al., BioTechniques 13 (1992)515-518; Li, M. Z., et al., Nature Methods 4 (2007) 251-256). Linearizedexpression plasmids coding for the rabbit kappa or gamma constant regionand VL of VH inserts were amplified by PCR using overlapping primers.Purified PCR products were incubated with T4 DNA-polymerase whichgenerated single-strand overhangs. The reaction was stopped by dCTPaddition. In the next step, plasmid and insert were combined andincubated with RecA which induced site specific recombination. Therecombined plasmids were transformed into E. coli. The next day thegrown colonies were picked and tested for correct recombined plasmid byplasmid preparation, restriction analysis and DNA-sequencing. Forantibody expression, the isolated HC and LC plasmids were transientlyco-transfected into HEK293 cells and the supernatants were harvestedafter 1 week. For cloning and expression of rabbit mouse chimericantibodies, the VH and VL regions were amplified by PCR and sub-clonedinto expression vectors containing the mouse constant kappa or mouseconstant gamma 1 region. The rabbit/mouse chimeric HC and LC plasmidswere isolated, tested by restriction analysis and DNA-sequencing forcorrect insertion and transiently co-transfected into HEK293 cells.Supernatants were harvested one week after transfection.

Antibody Purification

Recombinantly expressed rabbit antibodies were purified from cellculture supernatants on MabSelectSuRe™ columns (GE Healthcare). Prior tosample loading, the column was equilibrated with 25 mmol/L Tris-HCl, 25mmol/L NaCl, pH 7.4. Elution of the antibody was achieved with 50 mmol/Lacetate, pH 3.14. The eluted sample was immediately loaded onto adesalting column (Sephadex G25, GE Healthcare) and eluted in 20 mmol/LHis-HCl, 140 mmol/L NaCl, pH 6.0. This buffer was also used for thestorage of purified antibody. General storage temperature was 4° C.,room temperature during the purification process and −80° C. afteraliquotation. Recombinantly expressed rabbit/mouse chimaeras antibodiesfrom cell culture supernatants were purified on MabSelectSuRe™ columns(GE Healthcare). Prior to sample loading, the column was equilibratedwith 1×PBS, pH 7.4. Elution of the antibodies was achieved with 100mmol/L citrate, pH 3.0. The eluted sample was immediately neutralizedwith 2 mol/L Tris/HCl, pH 9.0. Afterwards the antibodies were loadedonto a size exclusion column (Superdex 200, GE Healthcare) and eluted in20 mmol/L His-HCl, 140 mmol/L NaCl, pH 6.0. This buffer was also usedfor the storage of purified antibodies. General storage temperature was4° C., room temperature during the purification process and −80° C.after aliquotation.

Example 2

Anti-Tau(pS422) monoclonal rabbit antibodies are highly selective fortau phosphorylated at pS422 and bind to fibrillary aggregates ofTau(pS422)

ELISA

Rabbit monoclonal antibodies were recombinantly expressed in HEK293cells. Cell culture supernatants or purified rabbit antibodies weretested for binding to biotinylated tau (416-430)[pS422],non-phosphorylated tau (416-430), KLH and the unrelated phospho-peptideMCAK_Human (88-102)[95-pSer] by ELISA. For preparation of ELISA plates,streptavidin pre-coated microtiter plates were incubated withbiotinylated tau (415-430)[pS422] at 50 ng/ml for 1 hour at roomtemperature. Coating with KLH or control peptides was performed at 1μg/ml. Rabbit anti-Tau(pS422) antibody (Abcam AB51071) or rabbitantibody containing supernatants were incubated in the antigen labeledmicrotiter plates for 60 minutes at various concentrations. Afterintensive washing, the binding of the rabbit antibodies was detectedusing a sheep anti-rabbit IgG digoxigenin conjugated detection antibody(Chemicon AQ301D). After incubation with TMB at room temperatureabsorbance at 370 nm-492 nm was measured. The antibody binding wascharacterized by its EC₅₀ values. Antibody binding to biotinylated tau(416-430)[pS422] and non-phosphorylated tau (416-430) peptides wascharacterized by its EC₅₀ values. Cross-reactivity with KLH or MCAKphosphopeptide was estimated by single-point measurement at highconcentrations, i.e., at 1:5 dilution of the cell culture supernatants.Results are shown in the Table below. EC₅₀ values of binding to tauphosphopeptide were found to be more than 100 times lower than EC₅₀values of binding to tau peptide, indicating at least 100 foldselectivity for phosphorylated tau fragment compared tonon-phosphorylated tau peptide.

Binding to KLH and MCAK control phosphopeptide was at background levelwith all antibodies, which is about 1 to 3% of the maximal valuemeasures with tau phosphopeptide.

TABLE EC₅₀ EC₅₀ non− phosphor- phosphor- OD 1:5 dilution ylated ylatedIgG titer of of supernatant tau peptide tau peptide supernatant KLH MCAK(μg/ml) (μg/ml) (μg/ml) (mE) (mE) mAb 005 <0.003 3.727 5.818 0.026 0.067mAb 019 <0.003 1.076 6.958 0.026 0.023 mAb 020 0.002 >3.369 3.369 0.0160.010 mAb 085 0.0009 0.146 6.46 0.029 0.062 mAb 086 0.0011 0.266 8.840.046 0.104 mAb 097 0.0013 1.281 19.87 0.042 0.029

Specificity for soluble and aggregated full-length Tau(pS422) was alsotested. Fibrillary aggregates of Tau(pS422) (300 μg/ml) were coated on apolystyrene based Maxisorb microtiter plate (Nunc) overnight at roomtemperature. In similar manner, soluble full-length tau and Tau(pS422)were coated on a Maxisorb microtiter plate. Rabbit anti-Tau(pS422)antibody control (Abcam AB51071), or purified rabbit antibodies wereadded and incubated for 60 minutes in concentrations up to 1000 ng/ml.After intensive washing, the binding of the rabbit antibodies wasdetected using a sheep anti-rabbit IgG digoxigenin conjugated detectionantibody (Chemicon AQ301D). After incubation with TMB at roomtemperature absorbance at 370 nm-492 nm was measured. The antibodybinding was characterized by its EC₅₀ values. Results are shown in thefollowing Table.

TABLE EC₅₀ fibrillary Rabbit EC₅₀ Tau(pS422) EC₅₀ tau Tau(pS422) mAbprotein (μg/ml) protein (μg/ml) (μg/ml) mAb 005 0.00034 no binding0.00755 mAb 019 0.00038 no binding 0.00059 mAb 020 0.00036 no binding0.00042 mAb 085 0.00025 no binding 0.00074 mAb 086 0.00023 no binding0.00048 mAb 097 0.00040 no binding 0.01358

Rabbit monoclonal antibodies bound to Tau(pS422) protein with EC₅₀values below 1 ng/ml. Fibrillary Tau(pS422) was detected with EC₅₀values ranging from 0.4 ng/ml to 14 ng/ml. Signals for binding tonon-phosphorylated full-lengths tau protein were indistinguishable frombackground levels. Therefore it was estimated that each of theantibodies binds to Tau(pS422) and fibrillary Tau(pS422) with aselectivity of at least 100-fold compared to tau.

BIAcore™

Binding to fibrillary Tau(pS422) aggregates was further investigated andconfirmed by BIAcore™ analysis. Measurements were performed using theBIAcore 3000 instrument at 37° C.

The system and sample buffer was HBS-EP (10 mM HEPES, 150 mM NaCl, 3.4mM EDTA, 0.005% Polysorbate 20 (v/v)). A BIAcore™ CMS sensor chip wassubjected to a preconditioning procedure. Sequentially 0.1% SDS, 50 mMNaOH, 10 mM HCl and 100 mM H₃PO₄ were injected for 30 seconds over theflow cells FC1, FC2, FC3 and FC4. The amine coupling procedure was doneaccording to the manufacturer's instructions using the BIAcore 3000™wizard v. 4.1. After an EDC/NHS activation of the sensor surface, anon-phosphoselective anti-tau antibody mAb <TAU>M-4/53-IgG wasimmobilized on sensor flow cells FC2, FC3 and FC4. As a control, anantibody against CK-MM (creatine kinase isotype), recognizing anirrelevant antigen, was captured on the FC1 flow cell. mAb<TAU>M-4/53-IgG and the antibody against CK-MM were diluted at 30 μg/mlin 10 mM NaAc, pH 5.0 and were injected at 10 μl/min for 7 minutescontact time to immobilize 10,000 RU of the antibody capturing system.The surface was deactivated by saturation with 1 M ethanolamine. Thesensor was conditioned by 5 cycles with phosphorylated filamentous tauprotein (stock 0.3 mg/ml diluted 1:100 in HBS-EP) as analyte in solutionat 10 μl/min for 2 minutes. Regeneration was performed with 10 mMglycine, pH 2.5 at 30 μl/min for 3 minutes. It was assumed, that theanalyte binding to mAb 4/53 does not dissociate the pTau filaments,because no dissociation of pTau filaments from the mAb 4/53 could beobserved. For all further measurement cycles, 0.3 mg/ml pTau filamentswere diluted 1:100 in HBS-EP buffer and were injected at 10 l/min for 1minute, in order to present pTau to the respective antibody analytes ina heterogeneous sandwich-mode. The antibody analytes were diluted inHBS-EP buffer to a concentration of 100 nM and were injected into thesystem at 20 l/min for 3 minutes. After 3 minutes of dissociation, thesensor surface was regenerated by 2 injections of a 10 mM glycine, pH2.5, for 1 minute at 100 l/min, followed by a HBS-wash for 15 seconds at100 μl/minute The association and dissociation phase of the interactionswere monitored. Since the antibody analyte in solution is bivalent, theavidity-burdened antibody-pTau kinetics were characterized by a biphasicdissociation model, consisting of a fast affinity-based earlydissociation step followed by an avidity-stabilized, but rate-limitingkinetic step in the latter complex dissociation. 10 seconds (early) and50 seconds (late) after analyte injection end, the kd and t/2(diss) werequantified, where possible. The kinetic measurements were evaluatedusing a double referencing procedure. First the signal from the FC1reference was subtracted to correct the buffer bulk effect andunspecific binding. Second the 0 nM analyte injection was subtracted tocorrect the dissociation of the primary antibodies from the respectivecapturing system. The kinetic rates were evaluated using a Langmuir 1.1dissociation fit model according to the BIAcore™ evaluation softwarev.4.1. The antigen/antibody complex stability halftime (min) wascalculated according to the formula ln(2)/60*kd.

Results are summarized in the following Table.

TABLE early (10s) late (50s) t/2diss t/2diss Clone kd (1/s) (min) kd(1/s) (min) mAb 005 2.19E−03 5.3 3.12 × 10⁻³  4 mAb 019 1.43E−02 0.86.17 × 10⁻⁴ 19 mAb 020 3.28E−03 3.5 4.08 × 10⁻⁴ 28 mAb 085 n.d. n.d.6.60 × 10⁻⁴ 18 mAb 086 1.62E−03 7.2 3.68 × 10⁻⁴ 32 mAb 097 n.d. n.d.n.d. n.d.

Example 3

Binding of anti-Tau(pS422) monoclonal rabbit antibodies to intracellularpTau in brain sections of Alzheimer's disease patients

The specific and sensitive immunohistochemical detection of pTaupathology in Alzheimer's disease brain tissue by monoclonal rabbitanti-Tau(pS422) antibodies was investigated by immunofluorescencestaining experiments using cryosections of human brain tissue from ADpatients. Rabbit IgGs were detected by goat anti-rabbit Alexa Fluor488®conjugated secondary antibodies (Invitrogen/Molecular Probes A11034).Specific and sensitive staining of pTau deposits and filaments wasevident for clones mAb 005, mAb 019, mAb 020, mAb 085, mAb 086 and mAb097. Intracellular pTau deposits, like large neurofibrillary tangles andelongated neutrophil threads, were noticeable. A minimal effectiveconcentration ranging between 0.08 and 0.016 μg/ml was determined forall clones investigated, which indicates highly sensitive binding togenuine human pTau deposits.

Example 4

Humanization of rabbit anti-human Tau(pS422) antibodies

The “variable domain” (variable domain of a light chain (VL), variabledomain of a heavy chain (VH)) as used herein denotes each of the pair oflight and heavy chain domains which are involved directly in binding theantibody to the Tau(pS422) antigen. The variable light and heavy chaindomains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions”.

The structures of the VH and the VL domain of the rabbit antibody mAb086 were analyzed in silico and compared to a structural database ofhuman VH and VL domains (IMGT). A panel of structurally most similar Vdomains was chosen for grafting the CDRs of the rabbit antibody onto thechosen human VH and VL domains. In addition, similarities in the primarysequence were taken into account to narrow down the choice of the humanV domains by aligning the primary sequence of the VH and VL domain ofthe rabbit antibody to the human V domain repertoire. Backmutationswithin the human framework regions to rabbit parent residues wereintroduced in some humanization variants. Similarly, mutations in theCDRs were introduced in some variants where appropriate to potentiallyincrease the affinity to the antigen, to maintain the CDR tertiarystructure, and to remove unwanted features like cysteine residues orresidues that can undergo modification after antibody purification.

The heavy and light chain vectors containing each of the humanizedvariant were co-transfected into HEK293 suspension cells in microtiterculture plates in a matrix manner to obtain cell cultures expressingfull size IgG of all possible light/heavy chain combinations. After 5days cultivation at 37° C., the supernatants were harvested and purifiedby protein A affinity chromatography in the microtiter scale.

Example 5

Generation of Recombinant Expression Vectors

a) Generation of vectors for the expression of immunoglobulin heavychains using the human IgG1 constant region

The humanized heavy chain encoding fusion gene comprising the human IgG1constant region (CH1, hinge, CH2, CH3) and a humanized anti-humanTau(pS422) antibody VH domain derived from rabbit antibody mAb 086 wasassembled by fusing a DNA fragment coding for the respective anti-humanTau(pS422)-specific antibody VH domain to a sequence element coding thehuman IgG1 constant region.

The human IgG1 constant region has the following amino acid sequence:

(SEQ ID NO: 58) ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVSWNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDELTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK.

The expression vector also comprised an origin of replication from thevector pUC18, which allows replication of this plasmid in E. coli, and abeta-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of the antibody heavy chain comprises thefollowing functional elements in 5′ to 3′ direction:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   a heavy chain variable (VH) domain encoding nucleic acid,    -   a human IgG1 constant region encoding nucleic acid, and    -   the bovine growth hormone polyadenylation sequence (BGH pA).        b) Generation of vectors for the expression of immunoglobulin        light chains using the human Ig-kappa constant region

The humanized kappa light chain encoding fusion gene comprising thehuman Ig-kappa constant region (CL-kappa) and an anti-human Tau(pS422)antibody VL (kappa) domain derived from rabbit antibody mAb 086 wasassembled by fusing a DNA fragment coding for the respective anti-humanTau(pS422) antibody VL (kappa) domain to a sequence element coding forthe human Ig-kappa constant region.

The human Ig-kappa constant region has the following amino acidsequence:

(SEQ ID NO: 59) RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYEKHKVYACEVT HQGLSSPVTK SFNRGEC.

The expression vector also comprised an origin of replication from thevector pUC18, which allows replication of this plasmid in E. coli, and abeta-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of the antibody kappa light chain comprises thefollowing functional elements in 5′ to 3′ direction:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   a light chain variable (VL) domain encoding nucleic acid,    -   a human Ig-kappa constant region encoding nucleic acid, and    -   the bovine growth hormone polyadenylation sequence (BGH pA).        c) Generation of vectors for the expression of immunoglobulin        light chains using the human Ig-lambda constant region

The humanized lambda light chain encoding fusion gene comprising thehuman Ig-lambda constant region (CL-lambda) and an anti-human Tau(pS422)antibody VL (lambda) domain derived from rabbit antibody mAb 086 wasassembled by fusing a DNA fragment coding for the respective anti-humanTau(pS422) antibody VL (lambda) domain to a sequence element coding forthe human Ig-lambda constant region.

The human Ig-lambda constant region has the following amino acidsequence:

(SEQ ID NO: 60) GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTVAWKADSSPVK AGVETTTPSK QSNNKYAASS YLSLTPEQWKSHRSYSCQVT HEGSTVEKTV APTECS.

The expression vector also comprised an origin of replication from thevector pUC18, which allows replication of this plasmid in E. coli, and abeta-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of the antibody lambda light chain comprises thefollowing functional elements in 5′ to 3′ direction:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   a variable light chain (VL) domain encoding nucleic acid,    -   a human Ig-lambda constant region encoding nucleic acid, and    -   the bovine growth hormone polyadenylation sequence (BGH pA).        d) Generation of Vectors for the Expression of Immunoglobulin        Kappa Light Chains Using the Human Ig-Kappa Constant Region

The human Ig-kappa light chain encoding fusion gene comprising the humanIg-kappa constant region (CL-kappa) and an anti-human Tau(S422) antibodyVL (kappa) domain derived from rabbit antibody mAb 086 was assembled byfusing a DNA fragment coding for the respective anti-humanTau(pS422)-antibody VL (kappa) domain to a sequence element coding forthe human Ig-kappa constant region. The construct was in a genomicorganization, i.e., introns were present in the signal peptide andbetween the VL (kappa) and the CL-kappa domains.

The expression vector also comprised an origin of replication from thevector pUC18, which allows replication of this plasmid in E. coli, and abeta-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of the antibody kappa light chain comprises thefollowing functional elements in 5′ to 3′ direction:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV),    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   a light chain variable (VL) domain encoding nucleic acid,    -   a human IgG kappa constant region, and    -   the bovine growth hormone polyadenylation sequence (BGH pA).        e) Generation of Vectors for the Expression of Immunoglobulin        Lambda Light Chains Using the Human Ig-Lambda Constant Region

The human Ig-lambda light chain encoding fusion gene comprising thehuman Ig-lambda constant region (CL-lambda) and an anti-human Tau(S422)antibody VL (lambda) domain derived from rabbit antibody mAb 086 wasassembled by fusing a DNA fragment coding for the respective anti-humanTau(pS422) antibody VL (lambda) domain to a sequence element coding forthe human Ig-lambda constant region. The construct was in a genomicorganization, i.e., introns were present in the signal peptide andbetween the VL (lambda) and the CL-lambda domains.

The expression vector also comprised an origin of replication from thevector pUC18, which allows replication of this plasmid in E. coli, and abeta-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of the antibody lambda light chain comprises thefollowing functional elements in 5′ to 3′ direction:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV),    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   a light chain variable (VL) domain encoding nucleic acid,    -   a human IgG lambda constant region, and    -   the bovine growth hormone polyadenylation sequence (BGH pA).

Example 6

Recombinant Production of Anti-Human Tau(pS422) Antibodies

Antibodies were produced in transiently transfected HEK293 cells (humanembryonic kidney cell line 293-derived), cultivated in F17 Medium(Invitrogen Corp.). For transfection of the respective vectors asdescribed in Example 5 “293-Free” Transfection Reagent (Novagen) wasused. The antibodies were expressed from individual expression plasmids.Transfections were performed as specified in the manufacturer'sinstructions. Recombinant antibody-containing cell culture supernatantswere harvested three to seven days after transfection. Supernatants werestored at reduced temperature (e.g., −80° C.) until purification.

General information regarding the recombinant expression of humanimmunoglobulins in, e.g., HEK293 cells is given in: Meissner, P. et al.,Biotechnol. Bioeng. 75 (2001) 197-203.

Example 7

Purification of Recombinant Anti-Human Tau(pS422) Antibodies

The antibody-containing culture supernatants were filtered and purifiedby two chromatographic steps.

The antibodies were captured by affinity chromatography using HiTrapMabSelectSuRe (GE Healthcare) equilibrated with PBS (1 mM KH₂PO₄, 10 mMNa₂HPO₄, 137 mM NaCl, 2.7 mM KCl), pH 7.4. Unbound proteins were removedby washing with equilibration buffer, and the antibody was recoveredwith 25 mM citrate buffer, pH 3.1, which was immediately after elutionadjusted to pH 6.0 with 1 M Tris-base, pH 9.0.

Size exclusion chromatography on Superdex 200™ (GE Healthcare) was usedas second purification step. The size exclusion chromatography wasperformed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The antibodycontaining solutions were concentrated with an Ultrafree-CL centrifugalfilter unit equipped with a Biomax-SK membrane (Millipore, Billerica,Mass., USA) and stored at −80° C.

Example 8

Kinetic Screening

Kinetic screening was performed according to Schraeml et al. (Schraeml,M. and M. Biehl, Methods Mol. Biol. 901 (2012) 171-181) on a BIAcore4000 instrument, mounted with a BIAcore CM5 sensor. The BIAcore 4000instrument was under the control of the software version V1.1. A BIAcoreCMS series S chip was mounted into the instrument and washydrodynamically addressed and preconditioned according to themanufacturer's instructions. The instrument buffer was HBS-EP buffer (10mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05% (w/v) P20). An antibodycapture system was prepared on the sensor surface. A polyclonal goatanti-human antibody with human IgG-Fc specificity (Jackson Lab.) wasimmobilized at 30 μg/ml in 10 mM sodium acetate buffer (pH 5) to spots1, 2, 4 and 5 in the instrument's flow cells 1, 2, 3 and 4 at 10,000 RUusing NHS/EDC chemistry. In each flow cell the antibodies were capturedon spot 1 and spot 5. Spot 2 and spot 4 were used as reference spots.The sensor was deactivated with a 1 M ethanolamine solution. Humanizedantibody derivatives were applied at concentrations between 44 nM and 70nM in instrument buffer supplemented with 1 mg/ml CMD(carboxymethyldextrane). The antibodies were injected at a flow rate of30 l/min for 2 minutes. The capture level (CL) of the surface-presentedantibodies was measured in rel. response units (RU). The analytes insolution, phosphorylated human tau protein, non-phosphorylated human tauprotein and the phosphorylated human tau mutant protein T422S, wereinjected at 300 nM for 3 minutes at a flow rate of 30 μl/minute Thedissociation was monitored for 5 minutes. The capture system wasregenerated by a 1 minute injection of 10 mM glycine buffer, pH 1.7 at30 μL/min over all flow cells. Two report points, the recorded signalshortly before the end of the analyte injection, denoted as binding late(BL) and the recorded signal shortly before the end of the dissociationtime, stability late (SL), were used to characterize the kineticscreening performance. Furthermore, the dissociation rate constant kd(1/s) was calculated according to a Langmuir model and theantibody/antigen complex half-life was calculated in minutes accordingto the formula ln(2)/(60*kd). The molar ratio (MR) was calculatedaccording to the formula MR=(Binding Late (RU))/(Capture level(RU))*(MW(antibody)/(MW(antigen)). In case the sensor was configuredwith a suitable amount of antibody ligand capture level, each antibodyshould be able to functionally bind at least to one analyte in solution,which is represented by a molar ratio of MR=1.0. Then, the molar ratiois also an indicator for the valence mode of analyte binding. Themaximum valence can be MR=2 for an antibody binding two analytes, onewith each Fab valence.

In another embodiment, kinetic rates were determined at 25° C. and 37°C. using the same experimental setup, but using multiple concentrationseries of each analyte in solution at 0 nM (buffer), 1.2 nM, 3.7 nM,11.1 nM, 33.3 nM, 100 nM and 300 nM. From the concentration-dependentbinding behavior the kinetic data was calculated using the BIAcoreevaluation software according to the manufacturer's instructions and aLangmuir 1.1 model with RMAX global.

Example 9

ELISA

Non-biotinylated peptide/protein/aggregate was added to non-coatedMaxisorb plates, biotinylated peptide/protein/aggregate in PBS was addedto streptavidin-coated Maxisorb plates, and plates were incubatedover-night. The supernatant was discarded and the wells washed threetimes with 90 μl wash buffer (1×PBS/0.1% Tween 20). Remaining reactivespots were blocked with blocking buffer (1×PBS/2% BSA (Bovine SerumAlbumin Fraction V, fatty acid free, Roche, Cat. No.: 10735078001)/0.05%Tween 20) by incubating for 1 hour. The supernatant was discarded andthe wells washed three times with 90 μl wash buffer. Samples and controlantibody were prepared in 12 dilutions (1:2) in ELISA buffer (1×PBS/0.5%BSA (Bovine Serum Albumin Fraction V, fatty acid free, Roche, Cat. No.:10735078001)/0.05% Tween 20) with a start concentration of 500 ng/mL.The incubation time was 60 minutes at room temperature on a shaker. Thesupernatant was discarded and the wells washed three times with 90 μlwash buffer. Solutions of secondary antibody were prepared in ELISAbuffer. A total of 25 μl antibody-mix was transferred to all wells ofthe assay plate and the plate was thereafter incubated on shaker for 60minutes at room temperature. The supernatant was discarded and the wellswere washed three times with 90 μl wash buffer. 25 μl of ABTS solutionwas added to all wells. The absorbance was read at 405 nm-492 nm.

Example 10

Kinetic Measurement of pTau Peptide Binding to Anti-pTau Antibodies(Capture Assay)

Binding of pTau fragment to anti-pTau antibodies was investigated bysurface plasmon resonance using a BIACORE T200 instrument (GEHealthcare). Around 10,000 resonance units (RU) of the capturing system(10 μg/ml goat anti-human Fc antibody; Jackson Immuno Research; OrderCode: JIR 109-005-098) were coupled on a CM5 chip (GE HealthcareBR-1005-30) at pH 5.0 by using an amine coupling kit supplied by the GEHealthcare. As running buffer, HBS-N pH 7.4 (10 mM HEPES, 150 mM NaCl,pH 7.4, GE Healthcare) was used. For measurement of the samples, runningand dilution buffer was PBS-T (10 mM phosphate buffered saline including0.05% Tween 20), pH 7.4. The sample block was set to 12° C. The flowcell was set to 25° C. and primed with running buffer twice.

The anti-pTau antibody was captured by injecting a 10 μg/ml solution for300 seconds at a flow of 10 μl/minute Association was measured byinjection of pTau fragment at various concentrations in solution for 180seconds at a flow of 30 μl/min starting with 300 nM in 1:3 dilutions.The dissociation phase was monitored for up to 300 seconds and triggeredby switching from the sample solution to running buffer. The surface wasregenerated by 60 seconds of washing with a 0.85% phosphoric acidsolution, followed by 60 seconds of washing with 5 mM sodium hydroxidesolution at a flow rate of 10 μl/minute Bulk refractive indexdifferences were corrected by subtracting the response obtained from agoat anti-human Fc antibody surface. Blank injections were alsosubtracted (=double referencing). For calculation of KD and otherkinetic parameters the Langmuir 1:1 model was used.

Example 11

Kinetic Measurement of Anti-pTau Antibody Fab Fragments Binding to pTauFragment (Direct Assay)

Binding of anti-pTau antibody Fab fragment samples to pTau fragment wasinvestigated by surface plasmon resonance using a BIACORE T200instrument (GE Healthcare). Approximately 20 resonance units (RU) ofbiotinylated pTau fragment (0.2 μg/ml;pTau(416-430)[Bi-XUUUU-416;pSer-422]amid) was coupled on a Series S SAchip (GE Healthcare BR-1005-31). Running and dilution buffer forimmobilization was HBS-N, pH 7.4 (10 mM HEPES, 150 mM NaCl, pH 7.4, GEHealthcare). For the following kinetic characterization, running anddilution buffer was PBS-T (10 mM phosphate buffered saline including0.05% Tween 20), pH 7.4. The sample block was set to 12° C. The flowcell was set to 25° C. and primed with running buffer twice.

Association was measured by injection of the anti-pTau antibody Fabfragment samples at various concentrations in solution for 180 secondsat a flow of 30 μl/min starting with 100 nM in 1:3 dilutions. Thedissociation phase was monitored for up to 600 seconds and triggered byswitching from the sample solution to running buffer. The surface wasregenerated by washing with one injection of 10 mM glycine solution, pH1.7 for 60 seconds at a flow rate of 10 μl/minute. Bulk refractive indexdifferences were corrected by subtracting the response obtained from ablank surface. Blank injections were also subtracted (=doublereferencing). For calculation of KD and other kinetic parameters theLangmuir 1:1 model was used.

Example 12

Kinetic Measurement of Anti-pTau Antibody Fab Fragments Binding to FullLength pTau (Direct Assay)

Binding of anti-pTau antibody Fab fragment samples to full length pTauprotein was investigated by surface plasmon resonance using a BIACORET200 instrument (GE Healthcare). Approximately 200 resonance units (RU)of biotinylated pTau protein (2 μg/ml) was coupled on a Series S SA chip(GE Healthcare BR-1005-31). Running and dilution buffer forimmobilization was HBS-N, pH 7.4 (10 mM HEPES, 150 mM NaCl, pH 7.4, GEHealthcare). For the following kinetic characterization, running anddilution buffer was PBS-T (10 mM phosphate buffered saline including0.05% Tween 20), pH 7.4. The sample block was set to 12° C. The flowcell was set to 25° C. and primed with running buffer twice.

Association was measured by injection of the anti-pTau antibody Fabfragment samples at various concentrations in solution for 180 secondsat a flow of 30 μl/min starting with 300 nM in 1:3 dilutions. Thedissociation phase was monitored for up to 600 seconds and triggered byswitching from the sample solution to running buffer. The surface wasregenerated by washing with one injection of 10 mM glycine solution, pH1.7 for 60 seconds at a flow rate of 10 μl/minute. Bulk refractive indexdifferences were corrected by subtracting the response obtained from ablank surface. Blank injections were also subtracted (=doublereferencing). For calculation of KD and other kinetic parameters theLangmuir 1:1 model was used.

Example 13

Effect of anti-Tau(pS422) antibody (VH35H5/VL31A1) and anti-Tau(pS422)antibody (VH35H5/VL49G1) on tau pathology in human tau-overexpressingmice

TauPS2APP transgenic mice overexpressing the longest human tau isoform(Grüninger et al., Neurobiol. Dis. 37 (2010) 294) were treated witheither with antibody 1 (VH35H5/VL31A1), antibody 2 (VH35H5/VL49G1) orvehicle for a period of 16 weeks. Just before treatment, all mice wereimmunosuppressed by administration of a single dose of an anti-CD4antibody (0.5 mg/mouse). The mice subsequently received weekly i.p.injections of the respective antibody or vehicle starting at 9 months ofage. For each of the two antibodies, three separate doses were tested,namely, 1.7 mg/kg, 5 mg/kg and 15 mg/kg. Mice were sacrificed 4-6 daysafter the last injection, transcardially perfused and the brains wereremoved for analysis. An untreated group of mice (9 months old) wassacrificed at the beginning of the experiment and used as baselinecomparator.

Each brain was sectioned sagittally and the individual hemispheressnap-frozen on dry ice. The left hemisphere was used for biochemicalquantitation of tau, Tau(pS422) and tau aggregates by immunoassay. Theright brain hemisphere was used for quantitative immunohistochemicalanalysis of Tau(pS422)-containing neurofibrillary tau.

Methods

Preparation of Brain Homogenates

One brain hemisphere from each mouse was weighed and added to 10 volumesof ice-cold extraction buffer (25 mM Tris-HCl, 800 mM NaCl, 10% sucrose,1 mM EGTA, pH 7.5, supplemented with 1% Protease Inhibitor Cocktail(Calbiochem #539134) and 1% Phosphatase Inhibitor Cocktail (Sigma#P0044)). The tissue was then homogenized in a 2 ml glass Douncehomogenizer. Homogenate was centrifuged at 20.000×g for 20 minutes at 4°C. Pelleted material was discarded, homogenate was aliquoted and storedat −20° C. until further use.

AlphaLISA Quantitation of Tau and Tau(pS422) in Brain Homogenates

AlphaLISA streptavidin-coated Donor and unconjugated Acceptor beads werepurchased from Perkin Elmer. Acceptor beads were coupled to eitheranti-tau antibody or anti-Tau(pS422) antibody.

Anti-tau antibody 5A6 (DSHB) was biotinylated on primary amine groupsusing biotin-N-hydroxysuccinamide (Thermo Fisher).

Tau measurement was performed in 1×HiBlock Assay Buffer (Perkin Elmer)using biotinylated-5A6 antibody, streptavidin Donor beads and anti-tauantibody Acceptor beads.

Tau(pS422) measurement was performed in Assay Buffer B (25 mM Hepes, pH7.4, 0.5% Triton X-100, 0.1% TopBlock (LuBioscience), 1 mg/ml Dextran500, 1/10 Blocking Reagent (Roche)) using biotinylated-5A6 antibody,streptavidin Donor beads and anti-Tau(pS422) antibody Acceptor beads.

Samples were prepared for assay in ½ AreaPlate-96 (Perkin Elmer) bymixing 5 μl of standard or sample with 10 μl of biotinylated-5A6antibody and 10 μl of antibody-coupled Acceptor beads then incubatingfor 1 hour at room temperature with shaking. The mixture was thensupplemented with 25 μl of streptavidin Donor beads and incubated for afurther 30 minutes at room temperature in the dark with shaking. Plateswere then measured in a Envision microtitre plate reader (Perkin Elmer)using laser excitation at 680 nm and a 615 nm emission filter. Standardcurves were generated using recombinant human tau (longest isoform) andERK-phosphorylated tau.

Quantitative immunohistochemical analysis of pTau pathology

Sagittal cryosections (thickness 10 μm) were prepared on a cryostat andfour sections per animal were immunofluorescence-stained with anantibody against Tau(5422) conjugated to goat anti-mouse/AlexaFluor555at a concentration of 10 μg/ml.

Image acquisition of entire brain sections was done with a Metafer 4slide scanning system (MetaSystems GmbH, Altlussheim, Germany). TheTau(pS422) immunopositive area was measured within the hippocampus andneocortical region and area of pTau-positive immunofluorescence signaldetermined by an unbiased morphometrical method by means ofcomputer-assisted image analysis. Quantification of pTau positive areawas done with Definiens image analysis software (Definiens AG, Munich,Germany).

Results

Treatment of TauPS2APP mice with either antibody 1 or antibody 2resulted in a dose-dependent reduction in Tau(pS422) accumulation inbrain homogenates (FIG. 7A-FIG. 7B). Using antibody 1 (VH35H5/VL31AS1),the reduction was statistically significant at 15 mg/kg. For antibody 2(VH35H5/VL49G1) significantly less Tau(pS422) was accumulated in alldose groups. The overall level of tau was unchanged in all dose groupsfor both antibodies (FIG. 8A-FIG. 8B). Quantitative immunohistochemicalanalysis to detect Tau(pS422)-containing neurofibrillary aggrgates wasperformed on the brains of antibody 1 mice (FIG. 9A-FIG. 9B). Althoughthere was no statistically significant lessening of tau pathology in anydose group, a trend to less pathology was observed with increasing dose.This was more apparent in the cortical region of the brains.

All documents cited in this application are hereby incorporated byreference as if recited in full herein.

Although illustrative embodiments of the present invention have beendescribed herein, it should be understood that the invention is notlimited to those described, and that various other changes ormodifications may be made by one skilled in the art without departingfrom the scope or spirit of the invention.

The invention claimed is:
 1. A humanized antibody that specificallybinds to human Tau(pS422), wherein the antibody comprises in the heavychain variable domain the HVRs of SEQ ID NO: 08, 77 and 10, and in thelight chain variable domain the HVRs of SEQ ID NO: 12, 14 and
 75. 2. Thehumanized antibody according to claim 1, comprising a heavy chainvariable domain of SEQ ID NO: 76 and a light chain variable domain ofSEQ ID NO:
 69. 3. The humanized antibody according to claim 1, whereinthe antibody is effector function silent.
 4. The humanized antibodyaccording to claim 1, wherein the antibody specifically binds to humanTau(pS422) (SEQ ID NO: 02) and does not bind to human Tau (SEQ ID NO:01).
 5. The humanized antibody according to claim 1, wherein theantibody is a) a full length antibody of the human subclass IgG 1; b) afull length antibody of the human subclass IgG4; c) a full lengthantibody of the human subclass IgG1 with the mutations L234A, L235A andP329G; d) a full length antibody of the human subclass IgG4 with themutations S228P, L235E and P329G; e) a full length antibody of the humansubclass IgG1 with the mutations L234A, L235A and P329G in both heavychains and the mutations T366W and S354C in one heavy chain and themutations T366S, L368A, Y407V and Y349C in the respective other heavychain; or f) a full length antibody of the human subclass IgG4 with themutations S228P, L235E and P329G in both heavy chains and the mutationsT366W and S354C in one heavy chain and the mutations T366S, L368A, Y407Vand Y349C in the respective other heavy chain.
 6. A humanized bispecificantibody, comprising: i) a first binding site comprising a heavy chainvariable domain of SEQ ID NO: 76 and a light chain variable domain ofSEQ ID NO: 69; and ii) a second binding site selected from: a) a heavychain variable domain of SEQ ID NO: 82 and a light chain variable domainof SEQ ID NO: 85, or b) a heavy chain variable domain of SEQ ID NO: 83and a light chain variable domain of SEQ ID NO: 85, or c) a heavy chainvariable domain of SEQ ID NO: 84 and a light chain variable domain ofSEQ ID NO: 85.